N-acetylcysteine amide (nac amide) for the treatment of diseases and conditions associated with oxidative stress

ABSTRACT

Methods and compositions comprising N-acetylcysteine amide (NAC amide) and derivatives thereof are used in treatments and therapies for human and non-human mammalian diseases, disorders, conditions and pathologies. Pharmaceutically or physiologically acceptable compositions of NAC amide or derivatives thereof are administered alone, or in combination with other suitable agents, to reduce, prevent, or counteract oxidative stress and free radical oxidant formation and overproduction in cells and tissues, as well as to provide a new source of glutathione.

FIELD OF THE INVENTION

The present invention generally relates to the treatment of mammalian,including human, diseases with antioxidants. More particularly, theinvention relates to treatments and therapies of a variety of diseasesand conditions involving the administration of N-acetylcysteine amide(NAC amide) or a derivative thereof, alone or in combination withanother agent, to a mammal in need thereof.

BACKGROUND OF THE INVENTION

Oxidative stress plays an important role in the progression ofneurodegenerative and age-related diseases, causing damage to proteins,DNA, and lipids. Low molecular weight, hydrophobic antioxidant compoundsare useful in treating conditions of peripheral tissues, such as acuterespiratory distress syndrome, amyotrophic lateral sclerosis,atherosclerotic cardiovascular disease, multiple organ dysfunctions andcentral nervous system neurodegenerative disorders, e.g., Parkinson'sdisease, Alzheimer's disease and Creutzfeldt-Jakob's disease. Oxidativestress has been causally linked to the pathogenesis of Parkinson'sdisease, Alzheimer's disease and Creutzfeldt-Jakob's disease, as well asother types of disorders. (U.S. Pat. No. 6,420,429 to D. Atlas et al.).

A deficiency of cellular antioxidants may lead to excess free radicals,which cause macromolecular breakdown, lipid peroxidation, buildup oftoxins and ultimately cell death. Because of the importance ofantioxidant compounds in preventing this cellular oxidation, naturalantioxidants, such as glutathione (GSH) (γ-glutamyl cysteinyl glycine)are continuously supplied to the tissues. GSH is synthesized by mostcells and is one of the primary cellular antioxidants responsible formaintaining the proper oxidation state within the body. When oxidized,GSH forms a dimer, GSSG, which may be recycled in organs producingglutathione reductase. In human adults, reduced GSH is produced fromGSSG, primarily in the liver, and to a smaller extent, by skeletalmuscle and red and white blood cells, and is distributed through theblood stream to other tissues in the body.

However, under certain conditions, the normal, physiologic supplies ofGSH are insufficient, its distribution is inadequate or local oxidativedemands are too high to prevent cellular oxidation. Under otherconditions, the production of and demand for cell antioxidants, such asGSH, are mismatched, thus leading to insufficient levels of thesemolecules in the body. In other cases, certain tissues or biologicalprocesses consume the antioxidants so that their intracellular levelsare suppressed. In either case, increased serum levels of antioxidant,e.g., glutathione, leads to increased amounts of the antioxidant thatcan be directed into cells. In facilitated transport systems forcellular uptake, the concentration gradient that drives uptake isincreased.

Glutathione N-acetylcysteine amide (NAC amide), the amide form ofN-acetylcysteine (NAC), is a low molecular weight thiol antioxidant anda Cu²⁺ chelator. NAC amide provides protective effects against celldamage. NAC amide was shown to inhibit tert.-butylhydroxyperoxide(BuOOH)-induced intracellular oxidation in red blood cells (RBCs) and toretard BuOOH-induced thiol depletion and hemoglobin oxidation in theRBCs. This restoration of thiol-depleted RBCs by externally applied NACamide was significantly greater than that found using NAC. Unlike NAC,NAC amide protected hemoglobin from oxidation. (L. Grinberg et al., FreeRadic Biol Med., 2005 Jan. 1, 38(1):136-45). In a cell-free system, NACamide was shown to react with oxidized glutathione (GSSG) to generatereduced glutathione (GSH). NAC amide readily permeates cell membranes,replenishes intracellular GSH, and, by incorporating into the cell'sredox machinery, protects the cell from oxidation. Because of itsneutral carboxyl group, NAC amide possesses enhanced properties oflipophilicity and cell permeability. (See, e.g., U.S. Pat. No. 5,874,468to D. Atlas et al.). NAC amide is also superior to NAC and GSH incrossing the cell membrane, as well as the blood-brain barrier.

NAC amide may function directly or indirectly in many importantbiological phenomena, including the synthesis of proteins and DNA,transport, enzyme activity, metabolism, and protection of cells fromfree-radical mediated damage. NAC amide is a potent cellular antioxidantresponsible for maintaining the proper oxidation state within the body.NAC amide can recycle oxidized biomolecules back to their active reducedforms and may be as effective, if not more effective, than GSH as anantioxidant.

Glutamate, an excitatory amino acid, is one of the majorneurotransmitters in the central nervous system (CNS). Elevated levelsof extracellular glutamate have been shown to be responsible for acuteneuronal damage as well as many CNS disorders, including hyperglycemia,ischemia, hypoxia (Choi, D. W., Neuron, 1(8):623-34, 1988), and chronicdisorders such as Huntington's, Alzheimer's, and Parkinson's diseases(Meldrum B. and Garthwaite J., Trends Pharmacol Sci., 11(9):379-87,1990; and Coyle J. T. and Puttfarcken P., Science, 262(5134):689-95,1993). Two mechanisms have been proposed for glutamate toxicity. Thefirst mechanism explains the excitotoxicity of glutamate as beingmediated through three types of excitatory amino acid receptors(Monaghan D. T. et al., Annu Rev Pharmacol Toxicol., 29:365-402, 1989).In addition to receptor-mediated glutamate excitotoxicity, it has alsobeen proposed that elevated levels of extracellular glutamate inhibitscystine uptake, which leads to a marked decrease in cellular GSH levels,resulting in the induction of oxidative stress (Murphy T. H. et al.,Neuron, 2(6):1547-58, 1989).

Cysteine is a critical component for intracellular GSH synthesis.Because of redox instability, almost all of the extracellular cysteineis present primarily in its oxidized state, cystine, which is taken upby cells via a cystine/glutamate transporter, the X c-system. Studiesindicate that glutamate and cystine share the same transporter;therefore, elevated levels of extracellular glutamate competitivelyinhibit cystine transport, which leads to depletion of intracellularGSH. (Bannai S, and Kitamura E., J Biol. Chem. 255(6):2372-6, 1980; andBannai S., Biochem Biophys Acta., 779(3):289-306, 1984). Depletion ofreduced glutathione results in decreased antioxidant capacity of thecell, accumulation of ROS (reactive oxygen species), and ultimatelyapoptotic cell death. Several studies have demonstrated the induction ofoxidative stress by glutamate in various cell lines including immaturecortical neurons (Murphy T. H. et al., FASEB J., 4(6):1624-33, 1990; andSagara J. et al., J. Neurochem., 61(5):1667-71, 1993), oligodendroglia(Oka A. et al., J. Neurosci., 13(4):1441-53, 1993), cultured ratastrocytes (Cho Y. and Bannai S., J. Neurochem., 55(6):2091-7, 1990),neuroblastoma cells (Murphy T. H. et al., Neuron., 2(6):1547-58, 1989),and PC12 cells (Froissard P. and Duval D., Neurochem Int., 24(5):485-93,1994).

Certain antioxidants such as NAC, lipoic acid (LA), (Han D. et al., AmJ. Physiol., 273:1771-8, 1997), tocopherol (Pereira C. M. and OliveiraC. R., Free Radic Biol Med., 23(4):637-47, 1997), and probucol (Naito M.et al., Neurosci Lett., 186(2-3):211-3, 1995) can protect againstglutamate cytotoxicity, mostly by replenishing GSH. However, in certainneurological diseases, such as cerebral ischemia and Parkinson'sdisease, enhancement of tissue GSH in brain regions cannot be attained,because these antioxidant agents have been obstructed by the blood-brainbarrier (Panigrahi M. et al., Brain Res., 717(1-2):184-8, 1996; and GotzM. E. et al., J Neural Transm Suppl., 29:241-9, 1990).

In addition to neurodegenerative diseases, such as those which affectthe brain and/or peripheral nervous tissues, other diseases, such asasthma, respiratory-related diseases and conditions, e.g., acuterespiratory distress syndrome (ARDS), amyotrophic lateral sclerosis (ALSor Lou Gerhig's disease), atherosclerotic cardiovascular disease andmultiple organ dysfunction, are related to the overproduction ofoxidants or reactive oxygen species by cells of the immune system.

A number of other disease states have been specifically associated withreductions in the levels of antioxidants such as GSH. Depressedantioxidant levels, either locally in particular organs or systemically,have been associated with a number of clinically defined diseases anddisease states, including HIV/AIDS, diabetes and macular degeneration,all of which progress because of excessive free radical reactions andinsufficient antioxidants. Other chronic conditions may also beassociated with antioxidant deficiency, oxidative stress, and freeradical formation, including heart failure and associated conditions andpathologies, coronary arterial restenosis following angioplasty,diabetes mellitus and macular degeneration.

Clinical and pre-clinical studies have demonstrated the linkage betweena range of free radical disorders and insufficient antioxidant levels.It has been reported that diabetic complications are the result ofhyperglycemic episodes that promote glycation of cellular enzymes andthereby inactivate the synthetic pathways of antioxidant compounds. Theresult is antioxidant deficiency in diabetics, which may be associatedwith the prevalence of cataracts, hypertension, occlusiveatherosclerosis, and susceptibility to infections in these patients.

High levels of antioxidants, such as GSH, have been demonstrated to benecessary for proper functioning of platelets, vascular endothelialcells, macrophages, cytotoxic T-lymphocytes, and other immune systemcomponents. Recently it has been discovered that patients infected withthe human immunodeficiency virus, HIV, exhibit low GSH levels in plasma,other body fluids, and in certain cell types, such as macrophages. Theselow GSH levels do not appear to be due to defects in GSH synthesis.Antioxidant deficiency has been implicated in the impaired survival ofpatients with HIV. (1997, PNAS USA, Vol. 94, pp. 1967-1972). Raisingantioxidant levels in cells is widely recognized as being important inHIV/AIDS and other disorders, because the low cellular antioxidantlevels in these disease types permit more and more free radicalreactions to fuel and exacerbate the disorders.

HIV is known to start pathologic free radical reactions, which lead tothe destruction of antioxidant molecules, as well as their exhaustionand the destruction of cellular organelles and macromolecules. Inmammalian cells, oxidative stresses, e.g. low intracellular levels ofreduced antioxidants and relatively high levels of free radicals,activate certain cytokines, including NF-κB and TNF-α, which, in turn,activate cellular transcription of the DNA to mRNA, resulting intranslation of the mRNA to a polypeptide sequence. In a virus-infectedcell, the viral genome is transcribed, resulting in viral RNAproduction, generally necessary for viral replication of RNA viruses andretroviruses. These processes require a relatively oxidized state of thecell, a condition which results from stress, low antioxidant levels, orthe production of reduced cellular products. The mechanism whichactivates cellular transcription is evolutionarily highly conserved, andtherefore it is unlikely that a set of mutations would escape thisprocess, or that an organism in which mutated enzyme and receptor geneproducts in this pathway would be well adapted for survival. Thus, bymaintaining a relatively reduced state of the cell (redox potential),viral transcription, a necessary step in late stage viral replication,is impeded.

The amplification effect of oxidative intracellular conditions on viralreplication is compounded by the actions of various viruses and viralproducts, which degrade antioxidants, such as GSH. For example, gp120,an HIV surface glycoprotein having a large number of disulfide bonds, isnormally present on the surface of infected cells. gp120 oxidizes GSH,resulting in reduced intracellular GSH levels. On the other hand, GSHreduces the disulfide bonds of gp120, thus reducing or eliminating itsbiological activity that is necessary for viral infectivity.Antioxidants such as GSH therefore interfere with the production of suchoxidized proteins and degrade them once formed. In a cell that isactively replicating viral gene products, a cascade of events may occurwhich can allow the cell to pass from a relatively quiescent stage withlow viral activity to an active stage with massive viral replication andcell death. This is accompanied by a change in redox potential. Bymaintaining adequate levels of antioxidant, this cascade may be impeded.

HIV is transmitted through two predominant routes, namely, contaminatedblood and/or sexual intercourse. In pediatric cases, approximately onehalf of the newborn individuals are infected in utero and one half areinfected at delivery. This circumstance permits a study of prevention oftransmission since the time of spread is known. Initially, there is anintense viral infection simulating a severe case of the flu, withmassive replication of the virus. Within weeks, this acute phase passesspontaneously as the body mounts a largely successful immune defense.Thereafter, the individual has no outward manifestations of theinfection. However, the virus continues to replicate within immunesystem cells and tissues, e.g., lymph nodes, lymphoid nodules,macrophages and certain multidendritic cells that are found in variousbody cavities.

Such stealthy and widespread infection is not just a viral problem. Thevirus, in addition to replicating, causes excessive production ofvarious free radicals and various cytokines in toxic or elevated levels.The cytokines are normally occurring biochemical substances that signalnumerous reactions and that typically exist in minuscule concentrations.Eventually, after an average of 7-10 years of seemingly quiescent HIVinfection, the corrosive free radicals and the toxic levels of cytokinesbegin to cause outward symptoms in infected individuals and failures inthe immune system begin. Substances like 15-HPETE are immunosuppressiveand TNF-α causes muscle wasting, among other toxic factors. The numbersof viral particles increase and the patient develops the Acquired ImmuneDeficiency Syndrome, AIDS, which may last 2 to 4 years before theindividual's demise. AIDS, therefore, is not merely a virus infection,although the viral infection is believed to be an integral part of theetiology of the disease.

Further, HIV has a powerful ability to mutate. It is this capabilitythat makes it difficult to create a vaccine or to develop long-term,antiviral pharmaceutical treatments. As more people fail to besuccessfully treated by the present complex regimens, the number ofresistant viral strains is increasing. Resistant strains of HIV are aparticularly dangerous population of the virus and pose a considerablehealth threat. These resistant HIV mutants also add to the difficultiesin developing vaccines that will be able to inhibit the activity ofhighly virulent viral types. Further, the continuing production of freeradicals and cytokines that may become largely independent of the virusperpetuate the dysfunctions of the immune system, the gastrointestinaltract, the nervous system, and many other organs in patients with AIDS.The published scientific literature indicates that many of these diverseorgan system dysfunctions are due to systemic deficiencies ofantioxidant compounds that are engendered by the virus and its freeradicals. For example, GSH is consumed in HIV infections because it isthe principal, bulwark antioxidant versus free radicals. An additionalcause of erosion of GSH levels is the presence of numerous disulfidebonds in HIV proteins, such as the gp120 cell surface protein. Disulfidebonds react with GSH and oxidize it. Thus, there is a need for otherantioxidants to be used to replace antioxidants such as GSH whose normalfunction is adversely affected by HIV infection.

The current HIV/AIDS pharmaceuticals take good advantage of the conceptof pharmaceutical synergism, wherein two different targets in oneprocess are affected simultaneously. The effect is more than additive.The drugs now in use were selected to inhibit two very different pointsin the long path of viral replication. The pathway of viral replicationas understood by skilled practitioners in the art is described in U.S.Pat. No. 6,420,429. New anti-HIV/AIDS therapies include additional drugsin the classes of Reverse Transcriptase inhibitors and proteaseinhibitors. Also, drugs are in development to block the integrase enzymeof the virus, which integrates the HIV DNA into the infected cell's DNA,analogous to splicing a small length of wire into a longer wire. Vaccinedevelopment also continues, although prospects seem poor because HIVappears to be a moving target and seems to change rapidly. Vaccinedevelopment is also impaired by the immune cell affinity of the virus.

Individuals infected with HIV have lowered levels of serum acid-solublethiols and antioxidants such as GSH in plasma, peripheral bloodmonocytes and lung epithelial lining fluid. In addition, it has beenshown that CD4⁺ and CD8⁺ T cells with high intracellular GSH levels areselectively lost as HIV infection progresses. This deficiency maypotentiate HIV replication and accelerate disease progression,especially in individuals with increased concentrations of inflammatorycytokines, because such cytokines stimulate HIV replication moreefficiently in cells in which antioxidant compounds are depleted. Inaddition, the depletion of antioxidants, such as GSH, is also associatedwith a process known as apoptosis, or programmed cell death. Thus,intercellular processes which artificially deplete GSH may lead to celldeath, even if the process itself is not lethal.

Diabetes mellitus (“diabetes”) is found in two forms: childhood orautoimmune (Type I, IDDM) and late-onset or non-insulin dependent (TypeII, NIDDM). Type I constitutes about 30% of the cases of diabetes. Therest of the cases are represented by Type II. In general, the onset ofdiabetes is sudden for Type I and insidious or chronic for Type II.Symptoms include excessive urination, hunger and thirst, with a slow andsteady loss of weight associated with Type I. Obesity is oftenassociated with Type II and has been thought to be a causal factor insusceptible individuals. Blood sugar is often high and there is frequentspilling of sugar in the urine. If the condition goes untreated, thevictim may develop ketoacidosis with a foul-smelling breath similar tosome who has been drinking alcohol. The immediate medical complicationsof untreated diabetes can include nervous system symptoms, and evendiabetic coma.

Because of the continuous and pernicious occurrence of hyperglucosemia(very high blood sugar levels), a non-enzymatic chemical reaction,called glycation, frequently occurs inside cells and causes a chronicinactivation of essential enzymes. One of the most critical enzymes,γ-glutamyl-cysteine synthetase, is glycated and readily inactivated.This enzyme is involved in a critical step in the biosynthesis ofglutathione in the liver. The net result of this particular glycation isa deficiency in the production of GSH in diabetics.

GSH is in high demand throughout the body for multiple, essentialfunctions, for example, within all mitochondria, to produce chemicalenergy called ATP. With a deficiency or absence of GSH, brain cells,heart cells, nerve cells, blood cells and many other cell types are notable to function properly and can be destroyed through apoptosisassociated with oxidative stress and free radical formation. GSH is themajor antioxidant in the human body and the only one that can besynthesized de novo. It is also the most common small molecular weightthiol in both plants and animals. Without GSH the immune system cannotfunction, and the central and peripheral nervous systems become aberrantand then cease to function. Because of the dependence on GSH as thecarrier of nitric oxide, a vasodilator responsible for control ofvascular tone, the cardiovascular system does not function well andeventually fails. Since all epithelial cells seem to require GSH,without GSH, intestinal lining cells also do not function properly andvaluable micronutrients are lost, nutrition is compromised, and microbesare given portals of entry to cause infections.

In diabetes, the use of GSH precursors cannot help to control GSHdeficiency due to the destruction of the rate-limiting enzyme byglycation. As GSH deficiency becomes more profound, the well-knownsequelae of diabetes progress in severity. The complications thatdevelop in diabetics are essentially due to runaway free radical damagesince the available GSH supplies in diabetics are insufficient. Forexample, a diabetic individual becomes more susceptible to infectionsbecause the immune system approaches collapse when GSH levels fall,analogous to the situation in HIV/AIDS. In addition, peripheralvasculature becomes comprised and blood supply to the extremities isseverely diminished because GSH is not available in sufficient amountsto stabilize nitric oxide to effectively exert its vascular dilation(relaxation) property. Gangrene is a common sequel and successiveamputations often result in later years. Peripheral neuropathies, theloss of sensation commonly of the feet and lower extremities develop andare often followed by aberrant sensations like uncontrollable burning oritching. Retinopathy and nephropathy are later events that are actuallydue to icroangiopathy, i.e., excessive budding and growth of new bloodvessels and capillaries, which often will bleed due to weakness of thenew vessel walls. This bleeding causes damage to the retina and kidneyswith resulting blindness and renal shutdown, which requires dialysistreatment. Further, cataracts occur with increasing frequency as the GSHdeficiency deepens. Large and medium sized arteries become sites ofaccelerated severe atherosclerosis, with myocardial infarcts at earlyages, and of a more severe degree. If coronary angioplasty is used totreat the severe atherosclerosis, diabetics are much more likely to havere-narrowing of cardiac vessels, termed restenosis.

Macular degeneration as a cause of blindness is a looming problem as thepopulation ages. Age-related macular degeneration (ARMD) ischaracterized by either a slow (dry form) or rapid (wet form) onset ofdestruction and irrevocable loss of rods and cones in the macula of theeye. The macula is the approximate center of the retina wherein the lensof the eye focuses its most intense light. The visual cells, known asthe rods and cones, are an outgrowth and active part of the centralnervous system. They are responsible and essential for the fine visualdiscrimination required to see clear details such as faces and facialexpression, reading, driving, operation of machinery and electricalequipment and general recognition of surroundings. Ultimately, thedestruction of the rods and cones leads to functional, legal blindness.Since there is no overt pain associated with the condition, the firstwarnings of onset are usually noticeable loss of visual acuity. This mayalready signal late stage events. It is now thought that one of the veryfirst events in this pathologic process is the formation of a materialcalled “drusen”, which first appears as either patches or diffuse dropsof yellow material deposited upon the surface of the retina in themacula lutea or yellow spot. This is the area of the retina wheresunlight is focused by the lens and which contains the highest densityof rods for acuity. Although cones, which detect color, are lost as wellin this disease, it is believed to be loss of rods, which causes theblindness. Drusen has been chemically analyzed and found to be composedof a mixture of lipids that are peroxidized by free radical reactions.

It is believed that the loss of retinal pigmented epithelial (RPE) cellsoccurs first in ARMD. Once an area of the retinal macula is devoid ofRPE cells, loss of rods, and eventually some cones, occurs. Finally,budding of capillaries begins and typical microangiopathy associatedwith late stage ARMD occurs. It is also known that RPE cells requirelarge quantities of GSH for their proper functioning. When GSH levelsdrop severely in cultures of RPE cells, the RPE cells begin to die. Whencultures of these cells are supplemented with GSH in the medium, theythrive. There is increasing evidence that progression of the disease ispaced by a more profound deficiency in GSH within the retina andprobably within these cells, as indicated by cell culture studies.

It is generally believed that “near” ultraviolet (UVB) and visual lightof high intensity primarily from sunlight is a strong contributingfactor of ARMD. People with light-colored irises constitute a high riskpopulation for macular degeneration, as do those with jobs that keepthem outdoors and those in equatorial areas where sunlight is mostintense. Additional free radical insults, e.g., smoking, adds to therisk of developing ARMD. Several approaches have been unsuccessfullytested to combat ARMD, including chemotherapy. Currently, there is noeffective therapy to treat ARMD. Laser therapy has been developed whichhas been used widely to slow the damage produced in the slow onset formof the disease by cauterizing neovascular growth. However the eventualoutcome of the disease, once it has started to progress, is certain.

The importance of thiols and especially of GSH to lymphocyte functionhas been known for many years. Adequate concentrations of GSH arerequired for mixed lymphocyte reactions, T-cell proliferation, T- andB-cell differentiation, cytotoxic T-cell activity, and natural killercell activity. Adequate GSH levels have been shown to be necessary formicrotubule polymerization in neutrophils. Intraperitoneallyadministered GSH augments the activation of cytotoxic T-lymphocytes inmice, and dietary GSH was found to improve the splenic status of GSH inaging mice, and to enhance T-cell mediated immune responses. Thepresence of macrophages can cause a substantial increase of theintracellular GSH levels of activated lymphocytes in their vicinity.Macrophages consume cystine via a strong membrane transport system, andgenerate large amounts of cysteine, which they release into theextracellular space. It has been demonstrated that macrophage GSH levels(and therefore cysteine equivalents) can be augmented by exogenous GSH.T-cells cannot produce their own cysteine, and it is required by T-cellsas the rate-limiting precursor of GSH synthesis. The intracellular GSHlevel and the DNA synthesis activity in mitogenically-stimulatedlymphocytes are strongly increased by exogenous cysteine, but notcystine. In T-cells, the membrane transport activity for cystine isten-fold lower than that for cysteine. As a consequence, T-cells have alow baseline supply of cysteine, even under healthy physiologicalconditions. The cysteine supply function of the macrophages is animportant part of the mechanism which enables T-cells to shift from aGSH-poor to a GSH-rich state.

The importance of the intracellular GSH concentration for the activationof T-cells is well established. It has been reported that GSH levels inT-cells rise after treatment with GSH; it is unclear whether thisincrease is due to uptake of the intact GSH or via extracellularbreakdown, transport of breakdown products, and subsequent intracellularGSH synthesis. Decreasing GSH by 10-40% can completely inhibit T-cellactivation in vitro. Depletion of intracellular GSH has been shown toinhibit the mitogenically-induced nuclear size transformation in theearly phase of the response. Cysteine and GSH depletion also affects thefunction of activated T-cells, such as cycling T-cell clones andactivated cytotoxic T-lymphocyte precursor cells in the late phase ofthe allogeneic mixed lymphocyte culture. DNA synthesis and proteinsynthesis in IL-2 dependent T-cell clones, as well as the continuedgrowth of preactivated CTL precursor cells and/or their functionaldifferentiation into cytotoxic effector cells are strongly sensitive toGSH depletion.

Glutathione status is a major determinant of protection againstoxidative injury. GSH acts on the one hand by reducing hydrogen peroxideand organic hydroperoxides in reactions catalyzed by glutathioneperoxidases, and on the other hand by conjugating with electrophilicxenobiotic intermediates capable of inducing oxidant stress. Theepithelial cells of the renal tubule have a high concentration of GSH,no doubt because the kidneys function in toxin and waste elimination,and the epithelium of the renal tubule is exposed to a variety of toxiccompounds. GSH, transported into cells from the extracellular medium,substantially protects isolated cells from intestine and lung againstt-butylhydroperoxide, menadione or paraquat-induced toxicity. Isolatedkidney cells also transport GSH, which can supplement endogenoussynthesis of GSH to protect against oxidant injury. Hepatic GSH contenthas also been reported to increase (i.e. to double) in the presence ofexogenous GSH. This may be due either to direct transport, as has beenreported for intestinal and alveolar cells, or via extracellulardegradation, transport, and intracellular resynthesis.

The nucleophilic sulfur atom of the cysteine moiety of GSH serves as amechanism to protect cells from harmful effects induced by toxicelectrophiles. It is well established that glutathione S-conjugatebiosynthesis is an important mechanism of drug and chemicaldetoxification. GSH conjugation of a substrate generally requires bothGSH and glutathione-S-transferase activity. The existence of multipleglutathione-S-transferases with specific, but also overlapping,substrate specificities enables the enzyme system to handle a wide rangeof compounds. Several classes of compounds are believed to be convertedby glutathione conjugate formation to toxic metabolites. For example,halogenated alkenes, hydroquinones, and quinones have been shown to formtoxic metabolites via the formation of S-conjugates with GSH. The kidneyis the main target organ for compounds metabolized by this pathway.Selective toxicity to the kidney is the result of the kidney's abilityto accumulate intermediates formed by the processing of S-conjugates inthe proximal tubular cells, and to bioactivate these intermediates totoxic metabolites.

The administration of morphine and related compounds to rats and miceresults in a loss of up to approximately 50% of hepatic GSH. Morphine isknown to be biotransformed into morphinone, a highly hepatotoxiccompound, which is 9 times more toxic than morphine in mouse bysubcutaneous injection, by morphine 6-dehydrogenase activity. Morphinonepossesses an α,β-unsaturated ketone, which allows it to form aglutathione S-conjugate. The formation of this conjugate correlates withloss of cellular GSH. This pathway represents the main detoxificationprocess for morphine. Pretreatment with GSH protects againstmorphine-induced lethality in the mouse.

The deleterious effects of methylmercury on mouse neuroblastoma cellsare largely prevented by co-administration of GSH. GSH may complex withmethylmercury, prevent its transport into the cell, and increasecellular antioxidant capabilities to prevent cell damage. Methylmercuryis believed to exert its deleterious effects on cellular microtubulesvia oxidation of tubulin sulfhydryls, and by alterations due toperoxidative injury. GSH also protects against poisoning by other heavymetals such as nickel and cadmium.

Because of its known role in renal detoxification and its low toxicity,GSH has been explored as an adjunct therapy for patients undergoingcancer chemotherapy with nephrotoxic agents such as cisplatin, in orderto reduce systemic toxicity. In one study, GSH was administeredintravenously to patients with advanced neoplastic disease, in twodivided doses of 2,500 mg, shortly before and after doses ofcyclophosphamide. GSH was well tolerated and did not produce unexpectedtoxicity. The lack of bladder damage, including microscopic hematuria,supports the protective role of this compound. Other studies have shownthat co-administration of GSH intravenously with cisplatin and/orcyclophosphamide combination therapy, reduces associated nephrotoxicity,while not unduly interfering with the desired cytotoxic effect of thesedrugs.

GSH has an extremely low toxicity, and oral LD₅₀ measurements aredifficult to perform due to the sheer mass of GSH, which has to beingested by the animal in order to see any toxic effects. GSH can betoxic, especially in cases of ascorbate deficiency, and these effectsmay be demonstrated in, for example, ascorbate deficient guinea pigsgiven 3.75 mmol/kg daily (1,152 mg/kg daily) in three divided doses,whereas in non-ascorbate deficient animals, toxicity was not seen atthis dose, but were seen at double this dose.

There is a need in the art for other compounds and therapeutic aspectsto treat a number of diseases that are linked to oxidative stress andthe presence of free oxygen radicals and associated disease pathogenesisin cells and tissues. Needed are antioxidant compounds, other than GSH,that are safe and even more potent, to overcome high oxidative stress inthe pathogenesis of diseases. Ideally, such compounds should readilycross the blood-brain barrier and easily permeate the cell membrane.Antioxidants such as vitamins E and C are not completely effective atdecreasing oxidative stress, particularly because, in the case ofvitamin E, they do not effectively cross through the cell membrane toreach the cytoplasm so as to provide antioxidant effects.

SUMMARY OF THE INVENTION

The present invention provides the use of a potent antioxidantN-acetylcysteine amide (NAC amide) or derivatives thereof, or aphysiologically acceptable derivative, salt, or ester thereof, in newapplications to treat disorders, conditions, pathologies and diseasesthat result from, or are associated with, the adverse effects ofoxidative stress and/or the production of free radicals in cells,tissues and organs of the body. NAC amide and its derivatives areprovided for use in methods and compositions for improving and treatingsuch disorders, conditions, pathologies and diseases.

As used herein, a “subject” within the context of the present inventionencompasses, without limitation, mammals, e.g., humans, domestic animalsand livestock including cats, dogs, cattle and horses. A “subject inneed thereof” is a subject having one or more manifestations ofdisorders, conditions, pathologies, and diseases as disclosed herein inwhich administration or introduction of NAC amide or its derivativeswould be considered beneficial by those of ordinary skill in the art.

In an aspect of the present invention, methods and compositionscomprising NAC amide provide an antioxidant to cells and tissues toreduce oxidative stress, and the adverse effects of cellular oxidation,in an organism. The invention provides a method of reducing oxidativestress associated with the conditions, diseases, pathologies asdescribed herein, by administering a pharmaceutically acceptableformulation of NAC amide or derivatives thereof to a human or non-humanmammal in an amount effective to reduce oxidative stress.

In another aspect of the present invention, NAC amide and itsderivatives are provided to treat an organism having a disorder,condition, pathology, or disease that is associated with theoverproduction of oxidants and/or oxygen free radical species. Accordingto this invention NAC amide treatment can be prophylactic ortherapeutic.

“Therapeutic treatment” or “therapeutic effect” means any improvement inthe condition of a subject treated by the methods of the presentinvention, including obtaining a preventative or prophylactic effect, orany alleviation of the severity of signs or symptoms of a disorder,condition, pathology, or disease or its sequelae, including those causedby other treatment methods (e.g., chemotherapy and radiation therapy),which can be detected by means of physical examination, laboratory, orinstrumental methods and considered statistically and/or clinicallysignificant by those skilled in the art.

“Prophylactic treatment” or “prophylactic effect” means prevention ofany worsening in the condition of a subject treated by the methods ofthe present invention, as well as prevention of any exacerbation of theseverity of signs and symptoms of a disorder, condition, pathology, ordisease or its sequelae, including those caused by other treatmentmethods (e.g., chemotherapy and radiation therapy), which can bedetected by means of physical examination, laboratory, or instrumentalmethods and considered statistically and/or clinically significant bythose skilled in the art.

In another aspect of the present invention, NAC amide is used in thetreatment and/or prevention of cosmetic conditions and dermatologicaldisorders of the skin, hair, nails, and mucosal surfaces when appliedtopically. In accordance with the invention, compositions for topicaladministration are provided that include (a) NAC amide, or derivativesthereof, or a suitable salt or ester thereof, or a physiologicallyacceptable composition containing NAC amide or its derivatives; and (b)a topically acceptable vehicle or carrier. The present invention alsoprovides a method for the treatment and/or prevention of cosmeticconditions and/or dermatological disorders that entails topicaladministration of NAC amide- or NAC-amide derivative-containingcompositions to an affected area of a patient.

In yet another of its aspects the present invention provides methods andcompositions useful for cancer and pre-cancer therapy utilizing NACamide or a derivative thereof, or its pharmaceutically acceptable saltsor esters. The present invention particularly relates to methods andcompositions comprising NAC amide or a derivative thereof in whichapoptosis is selectively induced in cells of cancers or precancers.

In another aspect, the present invention provides compositions andmethods comprising NAC amide or a derivative thereof for the suppressionof allograft rejection in recipients of allografts.

In another aspect, the present invention provides a NAC amide or aderivative thereof in a method of supporting or nurturing the growth ofstem cells for stem cell transplants, particularly stem cells culturedin vitro prior to introduction into a recipient animal, includinghumans.

In another aspect, the present invention provides methods of inhibiting,preventing, treating, or both preventing and treating, central nervoussystem (CNS) injury or disease, traumatic brain injury, neurotoxicity ormemory deficit in a subject, involving the administration of atherapeutically effective amount of NAC amide, or derivative thereof ora pharmaceutically acceptable composition thereof.

In another of its aspects, the present invention provides a method ofkilling or inhibiting the growth of microorganisms by providing NACamide in an amount effective to increase cellular levels of HIF-1 orHIF-1α to enhance the capacity of white blood cells to kill or inhibitthe growth of the microorganisms. Also in accordance with the invention,NAC amide is used as a countermeasure for biodefensive purposes, e.g.,in killing or growth inhibiting microorganisms, viruses, mycoplasma,etc., and in treating resulting diseases and conditions, as furtherdescribed herein.

In another aspect, the present invention provides a method of preventingtissue destruction resulting from the effects of metalloproteinases,such as MMP-3, which has been found to cause normal cells to express theRac1b protein, an unusual form of Rho GTPase that has previously beenfound only in cancers. Rac1b stimulates the production of highlyreactive oxygen species (ROS), which can promote cancer by activatingmajor genes that elicits massive tissue disorganization. In accordancewith the present invention NAC amide is used to block the effects ofRac1b-induced ROS production by administering or introducing NAC amideto cells, tissues, and/or the body of a subject in need thereof, totarget molecules in the pathways leading to tissue damage anddegradation. Thus, NAC amide can be used to inhibit MMP-3 and itsadverse functions, to target ROS indirectly or directly via theprocesses by which ROS activates genes to induce the EMT.

Another aspect of the present invention provides a method of stimulatingendogenous production of cytokines and hematopoietic factors, comprisingadministering or introducing NAC amide to cells, tissues, and/or asubject in need thereof for a period of time to stimulate the endogenousproduction. NAC amide can be used to stimulate production of cytokinesand hematopoietic factors, such as but not limited to, TNF-α, IFN-α,IFN-β, IFN-γ, IL-1, IL-2, IL-6, IL-10, erythropoietin, G-CSF, M-CSF, andGM-CSF, which are factors that modulate the immune system and whosebiological activities are involved in various human diseases, such asneoplastic and infectious diseases, as well as those involvinghematopoiesis and immune depressions of various origin (such as, withoutlimitation, erythroid, myeloid, or lymphoid suppression). Stimulation ofendogenous production of these cytokines and hematopoietic factors byNAC amide is particularly advantageous, since exogenous administrationof these cytokines and hematopoietic factors have limitations associatedwith the lack of acceptable formulations, their exhorbitant cost, shorthalf-life in biological media, difficulties in dose-determination, andnumerous toxic and allergic effects.

In another embodiment, the present invention encompasses methods andcomposition comprising NAC amide for detecting NAC-amide responsivechanges in gene expression in a cell, tissue, and/or a subject,comprising administering or introducing NAC amide or derivative of NACamide to the cell, tissue, and/or subject for a period of time to inducechanges in gene expression and detecting the changes in gene expression.NAC amide and derivatives thereof can induce changes in gene expressionsuch as genes involved in apoptosis, angiogenesis, chemotaxis, amongothers.

In another aspect, the present invention provides directed delivery ofNAC amide to cells, such as cancer cells that express high levels ofreceptors for folic acid (folate) or glutathione. According to thisaspect, NAC amide (“NACA”) is coupled to a ligand for the receptor(e.g., folic acid or glutathione) to form a conjugate, and then thisNACA-ligand conjugate is coated or adsorbed onto readily injectablenanoparticles using procedures known to those skilled in the art.According to this aspect, the nanoparticles containing NAC amide(“nano-NACA particles”) may be preferentially taken up by cancer ortumor cells where the NAC amide will exert its desired effects.Accordingly, the present invention provides a method of directeddelivery of NAC amide to host cells expressing high levels of surfacereceptor for a ligand, in which the method involves (a) coupling NACamide to the surface receptor ligand to form a NAC amide-ligandconjugate; (b) adsorbing the NAC amide-ligand conjugate ontonanoparticles; and (c) introducing the nanoparticles of (b) into thehost. The invention further provides a method of directed delivery ofNAC amide to host cells expressing high levels of surface receptor for aligand, in which the method involves (a) conjugating acetylateddendritic nanopolymers to a ligand; (b) coupling the conjugated ligandof (a) to NAC amide to form NAC amide-ligand nanoparticles; and c)introducing the nanoparticles of (b) into the host.

Another aspect of the present invention provides a compound of theformula I:

wherein:

-   -   R₁ is OH, SH, or S—S-Z;    -   X is C or N;    -   Y is NH₂, OH, CH₃—C═O, or NH—CH₃;    -   R₂ is absent, H, or ═O    -   R₃ is absent or

wherein: R₄ is NH or O;

-   -   R₅ is CF₃, NH₂, or CH₃

and wherein: Z is

with the proviso that if R₁ is S—S-Z, X and X′ are the same, Y and Y′are the same, R₂ and R₆ are the same, and R₃ and R₇ are the same.

The present invention also provides a NAC amide compound and NAC amidederivatives comprising the compounds disclosed herein.

In another aspect, a process for preparing an L- or D-isomer of thecompounds of the present invention are provided, comprising adding abase to L- or D-cystine diamide dihydrochloride to produce a firstmixture, and subsequently heating the first mixture under vacuum; addinga methanolic solution to the heated first mixture; acidifying themixture with alcoholic hydrogen chloride to obtain a first residue;dissolving the first residue in a first solution comprising methanolsaturated with ammonia; adding a second solution to the dissolved firstresidue to produce a second mixture; precipitating and washing thesecond mixture; filtering and drying the second mixture to obtain asecond residue; mixing the second residue with liquid ammonia and anethanolic solution of ammonium chloride to produce a third mixture; andfiltering and drying the third mixture, thereby preparing the L- orD-isomer compound.

In some embodiments, the process further comprises dissolving the L- orD-isomer compound in ether; adding to the dissolved L- or D-isomercompound an ethereal solution of lithium aluminum hydride, ethylacetate, and water to produce a fourth mixture; and filtering and dryingthe fourth mixture, thereby preparing the L- or D-isomer compound.

Another aspect of the invention provides a process for preparing an L-or D-isomer of the compounds disclosed herein, comprising mixingS-benzyl-L- or D-cysteine methyl ester hydrochloride or O-benzyl-L- orD-serine methyl ester hydrochloride with a base to produce a firstmixture; adding ether to the first mixture; filtering and concentratingthe first mixture; repeating steps (c) and (d), to obtain a firstresidue; adding ethyl acetate and a first solution to the first residueto produce a second mixture; filtering and drying the second mixture toproduce a second residue; mixing the second residue with liquid ammonia,sodium metal, and an ethanolic solution of ammonium chloride to producea third mixture; and filtering and drying the third mixture, therebypreparing the L- or D-isomer compound.

Yet another aspect of the invention provides a process for preparing acompound as disclosed herein, comprising mixing cystaminedihydrochloride with ammonia, water, sodium acetate, and aceticanhydride to produce a first mixture; allowing the first mixture toprecipitate; filtering and drying the first mixture to produce a firstresidue; mixing the second residue with liquid ammonia, sodium metal,and an ethanolic solution of ammonium chloride to produce a secondmixture; filtering and drying the second mixture, thereby preparing thecompound.

The present invention also provides a food additive comprising NAC amideor a NAC amide derivative as disclosed herein.

Additional aspects, features and advantages afforded by the presentinvention will be apparent from the detailed description andexemplification hereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A presents the structure of N acetyl cysteine. FIG. 1B presentsthe structure of N-acetylcysteine amide (NAC amide).

FIGS. 2A-2D show the cytotoxic response of PC12 cells to glutamate andprotection by NAC amide. PC12 cells were plated at a density 25×10³cells/well in a 24 well plate and grown for 24 h in culture medium. Theywere treated or not (control) with 10 mM Glu with or without NAC amide,as described in Example 1. Twenty-four hours later, cells were examinedand photographed. FIG. 2A: Control; FIG. 2B: NAC amide (NACA) only; FIG.2C: Glutamate only; FIG. 2D: Glutamate and NACA.

FIG. 3 shows the protective effect of NAC amide against glutamatecytotoxicity. Cells were plated and grown for 24 hours in a culturemedium; then they were treated or not (control) with 10 mM Glu, with orwithout NAC amide. Twenty-four hours later, the % LDH release wasdetermined using LDH analysis. Values represent means±SD. Statisticallydifferent values of * P<0.0001 and ** P<0.05 were determined, comparedto control. * P<0.0001 compared to glutamate-treated group.

FIG. 4 shows the effect of NAC amide on glutamate-induced cytotoxicity.Cells were exposed to 10 mM Glu, with or without NAC amide, for 24hours; the effects were compared to the control. Cell viability wasquantified by the MTS assay. Values represent means±SD. Statisticallydifferent values of *P<0.0005 and ** P<0.05 were determined, compared tocontrol. *** P<0.05 compared to glutamate-treated group.

FIG. 5 shows the effects of NAC amide [NAC amide] on cysteine levels inPC12 cells. Cells were plated and grown for 24 hours, and then they wereexposed to glutamate (10 mM) in the presence or absence of NAC amide(750 μM). Twenty-four hours later, the cells were harvested and cysteinelevels were measured. Values represent means±SD. Statistically differentvalues of * P<0.005 and ** P<0.05 were determined, compared to control.*** P<0.05 compared to glutamate-treated group.

FIG. 6 is a graph depicting a comparison of survival rates ofSprague-Dawley rats after X-ray irradiation treatment in combinationwith pre-treatment or post-treatment with NAC or NAC amide (TOVA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the use of an effective and potentantioxidant, glutathione N-acetylcysteine amide (NAC amide), (FIG. 1),or a physiologically or pharmaceutically acceptable derivative or saltor ester thereof, for use in a variety of disorders, conditions,pathologies and diseases in which oxidative stress and/or free radicalformation cause damage, frequently systemic damage, to cells, tissuesand organs of the body. The invention encompasses a pharmaceuticallyacceptable composition comprising NAC amide, e.g., water-soluble NACamide, or physiologically acceptable derivatives, salts, or estersthereof, which can be used in treatment and therapeutic methods inaccordance with this invention.

Glutathione N-acetylcysteine amide (NAC amide), the amide form ofN-acetylcysteine (NAC), is a novel low molecular weight thiolantioxidant and a Cu²⁺ chelator. NAC amide provides protective effectsagainst cell damage in its role as a scavenger of free radicals. Inmammalian red blood cells (RBCs), NAC amide has been shown to inhibittert.-butylhydroxyperoxide (BuOOH)-induced intracellular oxidation andto retard BuOOH-induced thiol depletion and hemoglobin oxidation in theRBCs. This restoration of thiol-depleted RBCs by externally applied NACamide was significantly greater than that found using NAC. Unlike NAC,NAC amide protected hemoglobin from oxidation. (L. Grinberg et al., FreeRadic Biol Med., 2005 Jan. 1, 38(1):136-45). In a cell-free system, NACamide was shown to react with oxidized glutathione (GSSG) to generatereduced glutathione (GSH). NAC amide readily permeates cell membranes,replenishes intracellular GSH, and, by incorporating into the cell'sredox machinery, protects the cell from oxidation. Because of itsneutral carboxyl group, NAC amide possesses enhanced properties oflipophilicity and cell permeability. (See, e.g., U.S. Pat. No. 5,874,468to D. Atlas et al.). NAC amide is also superior to NAC and GSH incrossing the cell membrane, as well as the blood-brain barrier. NACamide can be prepared as described in U.S. Pat. No. 6,420,429 to D.Atlas et al., the contents of which are incorporated by referenceherein.

NAC amide may function directly or indirectly in many importantbiological phenomena, including the synthesis of proteins and DNA,transport, enzyme activity, metabolism, and protection of cells fromfree-radical mediated damage. NAC amide is a potent cellular antioxidantresponsible for maintaining the proper oxidation state within cells. NACamide is synthesized by most cells and can recycle oxidized biomoleculesback to their active reduced forms. As an antioxidant, NAC amide may beas effective, if not more effective, than GSH.

In one embodiment, the present invention encompasses methods andcompositions comprising NAC amide for preventing, reducing, protecting,or alleviating glutamate-induced cytotoxicity in neurodegenerativediseases, particularly in neuronal cells and tissues (See, e.g., Example1). In this embodiment, NAC amide can protect cells of the nervoussystem from the effects of oxidative toxicity induced by glutamate.Without wishing to be bound by theory, NAC amide treatment can functionto supply GSH as a substrate for GSH peroxidase activity in affectedcells. In accordance with the present invention, NAC amide can inhibitlipid peroxidation, scavenge for reactive oxygen species (ROS) andenhance intracellular levels of GSH to combat and overcome oxidativestress. In addition, NAC amide can chelate lead and protect againstlead-induced oxidative stress. NAC amide is particularly beneficial andadvantageous for neurological disorders and diseases affecting the brainand associated parts thereof, because it more readily crosses theblood-brain barrier to enter the brain and provide its antioxidanteffects.

Different neurodegenerative conditions and diseases that can be treatedaccording to this embodiment include cerebral ischemia, Parkinson'sdisease. NAC amide can be used in the reduction of brain damage duringseizures; to provide resistance to induced epileptic seizures; forprotection during traumatic brain injury through the effect onmitochondrial function, reduction of inflammation and attenuation of andimprovement in re-profusion with decreased re-profusion injury; forreduction of traumatic brain injury; and for treating prion disease,such as Creutzfeldt-Jakob disease and mad cow disease, by acting as anNMDA receptor antagonist, by enhancing intracellular levels of theanti-apoptotic protein Bcl-2; and by increasing antioxidants toglutathione. NAC amide can be used in neural protection, mitochondrialpreservation and therapy potential after nerve injury, particularly toprevent primary sensory neuronal death.

In another embodiment, the invention embraces methods and compositionscomprising NAC amide for protecting cells and tissues fromradiation-induced oxidative stress. In accordance with this embodiment,NAC amide is superior to NAC in protecting tissues fromradiation-induced oxidative stress. (Example 2). The medical crisisfollowing the Chernobyl incident and the threat of a terrorist nuclearattack have raised awareness that high-dose total body irradiation mayoccur and result in death due to the induction of three potentiallylethal cerebrovascular, gastrointestinal and hematopoietic clinicalsyndromes, which result from high dose radiation exposure. Thecombination of the prodromal syndrome followed by the gastrointestinalsyndrome and bone marrow death induces dehydration, anemia, andinfection that lead to irreversible shock. Current treatment for thesubacute gastrointestinal and hematopoietic syndromes includessupportive therapy such as plasma volume expansion, platelets, andantibiotics to prevent dehydration and infection and promote bone marrowrepopulation. Human total body exposure to a radiation dose above 10 Gyhas been regarded as uniformly fatal. With therapeutic intervention,survival may be possible up to 15 Gy of total body irradiation, butbeyond 20 Gy the symptoms would not be manageable.

The systemic damage observed following irradiation is partially due tothe overproduction of reactive oxygen species (ROS), which disrupt thedelicate pro-oxidant/antioxidant balance of tissues leading to protein,lipid and DNA oxidation. For example, oxidation of the glucosaminesynthetase active site sulfhydryl groups is a key factor in the toxicityof the gastrointestinal syndrome. Polyunsaturated fatty acids, whenexposed to ROS, can also be oxidized to hydroperoxides that decompose inthe presence of metals to hydrocarbons and aldehydes such asmalondialdehyde (MDA). This lipid peroxidation can cause severeimpairment of membrane function through increased membrane permeabilityand membrane protein oxidation. DNA oxidation can lead to strandbreakage and consequent mutation or cell death. GSH is the principalintracellular thiol responsible for scavenging ROS and maintaining theoxidative balance in tissues, such as plasma, brain, kidney, liver andlung. In accordance with this embodiment, NAC amide significantlyimproves GSH levels in these tissues after radiation exposure. (Example2). The prevention of spinal cord damage resulting from radiationexposure is also encompassed by the use of NAC amide.

In another embodiment, the present invention encompasses methods andcompositions comprising NAC amide for stimulating endogenous productionof cytokines and hematopoietic factors, comprising administering orintroducing NAC amide to cells, tissues, and/or a subject in needthereof for a period of time to stimulate the endogenous production. NACamide can be used to stimulate production of cytokines and hematopoieticfactors, such as but not limited to, TNF-α, IFN-α, IFN-β, IFN-γ, IL-1,IL-2, IL-6, IL-10, erythropoietin, G-CSF, M-CSF, and GM-CSF, which arefactors that modulate the immune system and whose biological activitiesare involved in various human diseases, such as neoplastic andinfectious diseases, as well as those involving hematopoiesis and immunedepressions of various origin (such as, without limitation, erythroid,myeloid, or lymphoid suppression).

As used herein, “endogenous” means naturally occurring within a cell,tissue, or organism, or within a subject.

In another embodiment, the present invention encompasses methods andcomposition comprising NAC amide for detecting NAC-amide responsivechanges in gene expression in a cell, tissue, and/or a subject,comprising administering or introducing NAC amide or derivative of NACamide to the cell, tissue, and/or subject for a period of time to inducechanges in gene expression and detecting the changes in gene expression.The cell can be an endothelial cell, smooth muscle cell, immune cellsuch as erythroid, lymphoid, or myeloid cell, progenitors of erythroid,lymphoid, or myeloid cells, epithelial cell, fibroblasts, neuronal celland the like. The tissue can be any tissue of the subject, such as hair,skin, or nail tissue, vascular tissue, brain tissue, among many others.Preferably, the changes in gene expression are detected by microarrayanalysis, but other detection means can encompass, without limitation,reverse-transcription polymerase chain reaction (RT-PCR), NorthernBlotting, immunofluorescence, immunoblotting, or enzyme-linkedimmunosorbent assay, all of which are familiar techniques to thoseskilled in the art.

NAC amide and derivatives of NAC amide can induce changes in, forexample, endothelial cells that are indicative of an anti-angiogeniceffect. NAC has been shown to inhibit chemotaxis of endothelial cells inculture, and produce anti-angiogenic effects, such as modulation ofgenes responsible for blood vessel growth and differentiation, throughits antioxidant effects and upregulation of angiostatin (Pfeffer, U. etal, (2005) Mut. Res. 591: 198-211). Thus, NAC amide and NAC amidederivatives can be used to inhibit angiogenesis as an anti-cancer agent,for example, by preventing or inhibiting tumor growth and metastasis.

Cells, tissues, and/or a subject can be exposed to stimuli in thepresence of NAC amide or derivatives of NAC amide. Stimuli include, forexample, cells cultured in the presence of chemotactic orchemoattractant agents, like chemokines CXCL1-16, CCL1-27, XCL1, XCL2,RANTES, MIP 1-5 (alpha, beta, and gamma isoforms), MCP-1 through 5, andthe like. Cells, tissues, and subjects can also be stimulated withpharmaceutical agents, drugs, or treatment modalities. Afterstimulation, DNA, RNA, or protein can be isolated from the cells,tissues, and/or subject, and changes in gene expression can be detected.For example, total RNA can be isolated from cells according to standardtechniques known in the art and resultant cDNAs can be synthesized andsubsequently hybridized to a solid support, such as a silicon chip formicroarray analysis. Expression data and changes in the expression ofgenes in response to the stimuli can then be analyzed using computersoftware programs, such as GeneSpring (Silicon Genetics).

Non-limiting examples of such genes that exhibit changes in theirexpression include genes involved in or pertaining to cellular adhesion,apoptosis, chemokine and cytokine biosynthesis, synthesis ofextracellular matrix components, endothelium, inflammation, MAP kinases,metalloproteinases, NF-κB, nitric oxide, transforming growth factor(TGF) signaling, and blood vessels. Pfeffer et al reported that aplurality of NAC-responsive genes that are modulated (i.e., up- ordownregulated) include HSP40 (heat shock protein 40; DnaJ homolog),SERCA2 (Ca2+ transporting ATPase in cardiac muscle), MKP2 (MAP kinasephosphatase), TIP30 (HIV-1 Tat interactive protein 2), BTG1 (B-celltranslocation gene 1), TXL (thioredoxin-like), CRADD (Death receptoradaptor protein), WSX1 (Class I cytokine receptor), EMAP2 (endothelialmonocyte-activating protein), Jagged 1 (ligand for Notch receptor), MEA5(hyaluronoglucosaminidase), VRNA (Integrin αV), COL4A1 (Type IV collagenα1), uPA (urokinase plasminogen activator), CPE (carboxypeptidase E),TSPAN-6 (transmembrane 4 superfamily member 6), FGFB (basic fibroblastgrowth factor), I-TRAF (TRAF interacting factor), CDHH (cadherin 13),IL10RB (Interleukin-10 receptor β), MAP-1 (modulator of apoptosis 1),hCOX-2 (cyclooxygenase-2), CAS-L (Cas-like docking protein), CED-6(CED-6 protein), CX37 (gap junction protein α4), ABCG1 (ATP-bindingcassette protein, subfamily G), TRAIL (TNF ligand superfamily member10), and ESEL (endothelial adhesion molecule 1; Selectin E), as well asCHOP (DNA-damage-inducible transcript 3), PIM2 (pim-2 oncogene, MIF-1(homocysteine-inducible protein), PIG-A (phosphatidylinositol glycan,class A), K1AA0062, HK2 (hexokinase 2), UDPGDH (UDP-glucosedehydrogenase), ERF2 (Zinc finger protein 36, C3H type-like 2), RAMP(Zinc finger protein 198), Doc1 (Downregulated in ovarian cancer 1),GBP-1 (Guanylate-binding protein 1, interferon-inducible), GR(glucocorticoid receptor), ENH (LIM protein-enigma homolog), Id-2H(Inhibitor of DNA binding 2), BPGM (2,3-bisphosphoglycerate mutase),HOXA4 (Homeobox A10), EFNB2 (ephrin-B2), ART4 (Dombrock blood group),KIAA0740 (Rho-related BTB domain containing protein 1).

In another embodiment, the present invention encompasses methods andcompositions comprising NAC amide for stimulating macrophages andneutrophils to phagocytize infectious agents and other foreign bodiesand to eliminate microorganisms, mediated by reactive oxygen species andproteases. NAC amide can be used to improve macrophage function byincreasing glutathione availability, which, in turn, will improvealveolar function in fetal alcohol syndrome and to augment prematurealveolar macrophage function.

In another embodiment, the invention encompasses methods andcompositions comprising NAC amide to increase levels of intracellularreduced glutathione levels, which blocks the formation of irreversiblysickled cell red blood cells. Methods involving the administration ofNAC amide to prevent and treat sickle cell anemia and thalassemia areprovided.

In another embodiment, the invention encompasses methods andcompositions comprising NAC amide to treat leishmania through themechanism of histopathological modulation, in which cytokine pattern ismodified as demonstrated by a sustained higher frequency of interferon-γ(IFN-γ) and tumor necrosis factor alpha producing cells. NAC amide isused in the modulation of effector responses in animals, in conjunctionwith bi-glutathione.

In an embodiment, NAC amide is used to down-regulate cytokine synthesis,activation and downstream processes and/or to exert an antagonisticeffect on pro-inflammatory signals. Such an effect is beneficial in thetreatment of many diseases in which cytokines participate in thepathophysiology of the disease. For example, cytokines, which aremediators of oxidative stress, can alter the redox equilibrium byaffecting GSH/oxidized glutathione disulfide (GSSG) shuttling andrecycling. (For a review of the glutathione-mediated regulation ofcytokines and the role of antioxidants, see, J. J. Haddad, 2005, Mol.Immunol., 42(9):987-1014; and J. J. Haddad, 2002, Cellular Signalling,14(11):879-897). Additionally, liver injury related to theadministration of certain drugs can be initiated or intensified byinflammation states that stimulate unregulated production ofproinflammatory cytokines or growth factors, such as interferon γ, whichleads to the down-regulation of enzymes and proteins involved in drugmetabolism and elimination. NAC amide, or derivative thereof as an agentthat can decrease proinflammatory cytokine levels, is thus useful forpreventing and/or managing drug-induced hepatocytoxicity.

In another embodiment, the invention encompasses methods andcompositions comprising NAC amide or a derivative thereof for use as achemoprotectant against bone marrow toxicity after or duringchemotherapy, including alkylators with or without glutathionedepletion.

In another embodiment, the invention encompasses methods andcompositions comprising NAC amide or a derivative thereof to treatvarious aspects of sepsis, particularly bacterial sepsis and septicshock, including gram-negative septic shock. NAC amide and itsderivatives can act as an inhibitor of the nuclear factor NF-κB, whichprevents staphylococcal enterotoxin A (SCC) fever by acting through thehuman peripheral blood mononuclear cells to block the stimulation andsynthesis or release pyrogenic cytokines and to block inflammatorysponsors through the regulation of genes in coding for proinflammatorycytokines. In accordance with this embodiment, NAC amide or a derivativethereof is used to block lipid peroxidation and to improve the diseasestatus in children with acute purulent meningitis and encephalitis. NACamide and its derivatives can be used to block pertussis toxin secretionby Bordetella pertussis and for the treatment of lethal sepsis bylimiting inflammation and potentiating host defense. Because decreasedbacterial colonies improve survival, migration of neutrophils to thesite of infection and to a distant site is upregulated and optimal GSHlevels are important for an efficient response to sepsis. In addition,ROS release by immune cells are important mediators in sepsis and septicshock. During a normal immune response antioxidant serves todown-regulate the ongoing immune response mostly through modulation ofproinflammatory mediators.

In another embodiment, methods and compositions comprising NAC amide ora derivative thereof can be used in the treatment of infection anddisease caused by microorganisms and the like, such as bacteria,parasites, nematodes, yeast, fungi, plasmodia, mycoplasma, spores, andthe like, e.g., malarial infections and tuberculosis and rickettsiainfection. In a related aspect, it has recently been found thatinfection by a number of types of bacteria, such as Streptococcus,Staphylococcus, Salmonella, Bacillus (Tubercule bacillus) etc., whichcause diseases in humans, induce a direct response by leukocytes (i.e.,white blood cells) in the body, to increase their levels of hypoxiainducible transcription factor-1, or HIF-1. The HIF-1 protein binds tocellular DNA and activates specific genes to help cells function in alow oxygen environment. HIF-1, in turn, stimulates the white blood cellsto produce and release antimicrobial compounds, e.g., small proteins,enzymes and nitric oxide, that work together to kill bacteria. Inaddition, it has been found that low oxygen levels, which occur at thesite of an infection, activate HIF-1 in macrophages and neutrophils,which typically ingest and destroy invading microorganisms. The greaterthe increase in HIF-1 levels in the white blood cells, the greater theiranti-bacterial activity. In accordance with this aspect of the inventionand in view of the influence of HIF-1 in regulating the killingfunctions of white blood cells, an alternative to the direct killing ofbacteria, etc. is to use agents, e.g., small molecules, that promoteHIF-1 activity in white blood cells to boost their bacterial killingability, thereby promoting a resolution to infection through the actionsof the immune system's natural defense mechanisms. One such agent is NACamide, which can be used in a method of killing or inhibiting the growthof microorganisms by increasing cellular levels of HIF-1, i.e., HIF-1,thereby enhancing the capacity of white blood cells, such asmacrophages, to kill the microorganisms. Because N-acetyl-L-cysteine,NAC, a glutathione (GSH) precursor and a ROS scavenger, which does notpossess the enhanced properties of lipophilicity and cell permeabilityof NAC amide, has been shown to induce HIF-1α in epithelial cells (J. J.E. Haddad et al., 2000, J. Biol. Chem., 275:21130-21139), the use of NACamide to modulate HIF-1 α production in white blood cells in order toactivate the bacterial killing potential of these cells is embraced asan improved antioxidant treatment provided by the present invention. Thepresent invention is further directed to the use of NAC amide or aderivative thereof as a bacteriostatic agent when used as a treatmentfor bacterial infection, particularly antibiotic resistant, ormulti-antibiotic resistant bacteria such as tuberculosis-causingmicroorganisms.

In a related embodiment, the present invention is directed to the use ofNAC amide or a derivative thereof as a biodefensive agent for inducingthe killing of infecting or contaminating microorganisms. These types ofmicroorganisms may pose a severe health threat if they should bedisseminated to the public and/or genetically altered so as to beantibiotic resistant. The following lists set forth categories ofmicroorganisms, viruses, diseases and agents for which NAC amide or itsderivative is provided as a suitable countermeasure, used alone, or incombination with other active compounds, agents and substances to treataffected organisms and/or cells thereof:

Infectious Diseases: Aflatoxins, Alphavirus Eastern equine encephalitisvirus, Alphavirus Venezuelan equine encephalitis virus,Antibiotic-resistant Mycobacterium tuberculosis, Arenavirus Junin Virus,Arenavirus Lassa Virus, Ascaris lumbricoides (roundworm), Avianinfluenza, Bacillus anthracis (anthrax), Borrelia, Brucella,Burkholderia mallei (glanders), Chlamydia psittaci (parrot fever),Chlamydia trachiomitis (Trachoma), Clostridium botulinum (botulism),Clostridium perfringens (gas gangrene), Coccidioidomycosis immitis,Coxiella burnetti (Q fever), Cryptosporidium parvum, Dinoflagellateneurotoxin (Paralytic Shellfish Toxin), Drancunculus medianensis (guineaworm), Ebola virus, Entamoeba histolytica (amoebiasis), Epsilon toxin ofClostridium perfringens, Escherichia coli, Flavivirus Yellow Fever virus(e.g., West Nile virus, Dengue), Francisella tularensis (tularemia),Giardia lamblia (giardiasis), Hantavirus, Henipavirus Nipah virus (Nipaencephalitis), HIV and AIDS, Influenza, Leishmania donovane, Marburgvirus, Methicillin-resistant staphylococcus aureus (MRSA), Mycobacteriumleprea (leprosy), Mycobacterium ulcerans (Burulu ulcer), Nairo virusCrimean-Congo hemorrhagic fever virus, Necator Americanus/Ancylostomaduodenale (hookworm), Onchocerca volvulus (river blindness), Orthopoxvirus, Pathogenic Haemophilus, Pathogenic Salmonella, PathogenicShigella, Pathogenic Streptococcus, Phlebovirus Rift Valley fever virus,Plasmodium falciparum, P. ovale, P. vivax, P. malariae (malaria), Ricintoxin (castor bean oil), Rickettsia rickettsii (Rocky Mountain SpottedFever), Rickettsia typhi (typhus), Salmonella typhi (typhoid fever),Schistosoma mansoni, S. haematobium, S. japonicum, Shigella dysenteriae,Smallpox, Staphylococcus enterotoxin B, Tickborne encephalitis virus,Tickborne hemorrhagic fever viruses, Toxoplasma gondii, Treponema,Trichothecene Mycotoxins, Trichuris trichiura (whipworm), Trypanosomabrucei, T. gambiense or T. rhodesiense, Vibrio species (cholera),Wuchereria bancrofti and Brugia malayi, Yersinia pestis (black death).

Other Threats: Blister agents, including Lewisite, nitrogen and sulfurmustards; Blood agents, including hydrogen cyanide and cyanogenschloride; Exotic agents, including hybrid organisms, geneticallymodified organisms, antibiotic-induced toxins, autoimmune peptides,immune mimicry agents, binary bioweapons, stealth viruses andbioregulators and biomodulators; Heavy metals, including arsenic, leadand mercury; incapacitating agents, including BZ; nerve agents,including Tabun, Sarin, Soman, G F, VX, V-gas, third generation nerveagents, organophosphate pesticides and carbamate insecticides; nuclearand radiological materials, pulmonary agents, including phosgene andchorine vinyl chloride; volatile toxins, including benzene, chloroformand trihalomethanes. In accordance with the present invention, NAC amideor derivatives thereof can serve as an innovative treatment for knownand emerging natural infectious disease threats, as well as trauma,e.g., excessive bleeding and other events, associated with and/orresulting from an act of bioterrorism.

Illustratively, Rickettsia, which causes the pathogenesis of typhus andspotted fever rickettsioses, results in serious adverse vascular andhemorrhagic conditions, (e.g., increased vascular permeability andedema) notably in the brain and lung, following its entry into vascularendothelial cells. R. rickettsii-infected endothelial cells produce ROScausing peroxidative damage to cell membranes. (D. J. Silverman et al.,1990, Ann. N.Y. Acad. Sci., 590:111-117; D. H. Walker et al., 2003, Ann.N.Y. Acad. Sci., 990:1-11). Because the oxidative-stress mediated damageto R. rickettsii-infected endothelial cells is associated with thedepletion of host components such as GSH and levels of catalase that actas host defenses against ROS-induced damage, the concentration ofhydrogen peroxide and ROS increase in the cells to cause ROS-inducedcellular damage. In a similar manner, cells, e.g., fibroblasts that areinfected with Mycoplasma (e.g., Mycoplasma pneumoniae) also produceincreased intracellular levels of hydrogen peroxide and decreased levelsof catalase, resulting in oxidative stress that can lead to death of theinfected cells. (M. Almagor et al., 1986, Infect. Immun.,52(1):240-244). To provide an ameliorating effect of oxidative stressinduced in cells by infecting microorganisms such as Rickettsia,Mycoplasma, etc., NAC amide or a derivative thereof is provided to aninfected host as an antioxidant therapeutic. NAC amide administration tocells and/or organisms (e.g., infected host mammals) in accordance withthe present invention, alone or in combination with other agents and/orantioxidants, can limit the amount and/or extent of oxidative damagethat is induced by microbial infection.

In another embodiment, the invention encompasses methods andcompositions comprising NAC amide or a derivative thereof for use inpreventing periventricular leukomalacia (PVL). NAC amide or a derivativethereof may provide neural protection and attenuate the degeneration ofOPCs against LPS evoked inflammatory response in white matter injury indeveloping brain. Moreover, NAC amide or a derivative thereof may beused as a treatment for placental infection as a means of minimizing therisk of PVL and cerebral palsy (CP).

In another embodiment, the invention encompasses methods andcompositions comprising NAC amide or a derivative thereof for thetreatment of osteoporosis. The tumor necrosis factor member RANKLregulates the differentiation, activation and survival of osteoclaststhrough binding of its cognate receptor, RANK. RANK can interact withseveral TNF-receptor-associated factors (TRAFs) and activate signalingmolecules including Akt, NF-κB and MAPKs. Although the transientelevation of reactive oxygen species by receptor activation has beenshown to act as a cellular secondary messenger, the involvement of ROSin RANK signal pathways has not been characterized. RANKL can stimulateROS generation and osteoclasts. According to this embodiment, NAC amidecan be used to pretreat or treat osteoclasts so as to achieve areduction in RANKL-induced Akt, NF-κB, and ERK activation. The reducedNF-κB activity by NAC amide may be associated with decreased IKKactivity and IκBα phosphorylation. Pretreatment with NAC amide or aderivative thereof can be used to reduce RANKL-induced actin ringformation required for bone resorbing activity and osteoclast survival.The methods and compositions comprising NAC amide or a derivativethereof can be used for the improvement of osteoporosis through blockageand interference with osteoclasts, and to lower reactive oxidativestress levels so as to have beneficial effects on preventing bone lossby reducing RANKL-induced cellular function.

In a related embodiment, NAC amide or a derivative thereof is used inthe treatment of osteoporosis by blockage of thiol thioredoxin-1, whichmediates osteoclast stimulation by reactive oxidation species (ROS), aswell as blockage of TNF-α, which causes loss of bone, particularly incircumstances of estrogen deficiency.

In another embodiment, the invention embraces methods and compositionscomprising NAC amide or a derivative thereof are used for the treatmentof polycystic ovary syndrome. NAC amide or a derivative thereof may alsobe used as a therapeutic agent to ameliorate the homocysteine and lipidprofiles in PCOS-polycystic ovary syndrome.

In another embodiment, the invention encompasses the use of NAC amide ora derivative thereof in treatments and therapies for toxin exposure andconditions related thereto, e.g., sulfur mustard (HD-induced lunginjury). Treatment of individuals having been exposed to toxins orsuffering from toxin exposure with NAC amide or a derivative thereof mayreduce neutrophil counts to achieve a decreased inflammatory response.NAC amide and its derivatives may be useful as a treatment compound forpatients having sulfur mustard vapor exposure induced lung injury.Administration of NAC amide or a derivative thereof can be either orallyor as a bronchoalveolar lavage. As an agent having anti-glutamate toxinactivity, NAC amide and its derivatives are useful in methods andcompositions for the blockage of brain and/or lung damage and cognitivedysfunction in mechanical warfare agents including CW, vesicants, sulfurmustard, nitrogen mustards, chloroethyl amine, lewisite, nerve agentsO-ethyl S-(2-[di-isopropylamino]ethyl)methyl phosphorothioate (VX),tabun (GA) and sarin (GB) and soman DG and the blood agentscuianogenchloride, and in the prevention of organophosphate inducedconvulsions and neuropathological damage.

In another embodiment, the present invention encompasses methods andcompositions comprising NAC amide for use in the treatment of burntrauma. NAC amide or a derivative thereof can block NF-κB, which hasbeen shown to reduce burn and burn sepsis. NAC amide or a derivativethereof can be used to protect microvascular circulation, reduce tissuelipid peroxidation, improve cardiac output and reduce volume of requiredfluid resuscitation. NAC amide or a derivative thereof can be used inthe prevention of burn related cardiac NF-κB nuclear migration, andimprove cardiomyocyte secretion of TNF-α, IL-1β, and IL-6 and to improvecardiac malfunction. An association between cellular oxidative stressand burn-mediated injury provides an avenue for administering NAC amideor a derivative thereof as an antioxidant that can inhibit free radicalformation and/or scavenge free radicals to protect tissues and organs inpatients with burn injury.

In another embodiment, the present invention encompasses methods andcompositions comprising NAC amide or a derivative of NAC amide for usein the prevention of lung injury due to the adverse effects of airpollution and diesel exhaust particles.

In another embodiment, the present invention encompasses methods andcompositions comprising NAC amide or a derivative thereof for use in thetreatment and therapy of cardiovascular disease and conditions. NACamide and its derivatives can be used as a blocker ofangiotensin-converting enzyme. In acute myocardial infarction, NAC amideor a derivative thereof can be used to decrease oxidative stress, and tocause more rapid re-profusion, better left ventricular preservation,reduced infarct size, better preservation of global and regional leftventricular function and modification of QSR complex morphology and ECG.NAC amide or a derivative thereof can also be used in the treatment offocal cerebral ischemia with protection of the brain and reduction ofinflammation in experimental stroke. NAC amide can be used in thetreatment of reperfusion injuries, as well as apoptosis of myocardialendothelial cells and interstitial tissue. As a nutriceutical, NAC amideor a derivative thereof may assist in the elevation of nitric oxidelevels, play an important role in the management of cardiovasculardisease, reduce chronic inflammation in cardiovascular disease andprevent restenosis of cardiovascular stents placed in coronary arteriesand carotid arteries. NAC amide and its derivatives can be used in theprevention of cardiac failure following MI and cardiomyopathy due toprevention of oxidative stress and improvement of left ventricularremodeling. Use of NAC amide or a derivatives of NAC amide in thiscapacity supports the involvement of oxidative stress in myocardialvascular dysfunction and hypertension and provides a role forantioxidant strategies to preserve the myocardial microvasculature. NACamide or a derivative thereof can also be used in the prevention ofoxidized proteins in muscles.

In another embodiment of the present invention, method and compositionscomprising NAC amide or a derivative thereof can be used to treatarterial sclerosis and to increase high density lipoprotein(HDL)-cholesterol serum levels in hyperlipidemic and normal lipidemicindividuals with documented coronary stenosis. NAC amide or a derivativethereof can also be used to decrease coronary and alpha-beta stress; toprevent further myocardial infarctions; and to cause a reduction in bodyfat thereby improving glucose tolerance, particularly in overweight orobese individuals. NAC amide or a derivative thereof be used to improvemuscular performance and decrease levels of tumor necrosis factor in oldage.

In other embodiments, the present invention is directed to the use ofmethod and compositions comprising NAC amide or a derivative thereof inthe treatment of thalassemic blood by ameliorating oxidative stress inplatelets. The activation of platelets causes thromboembolicconsequences and produces a hypercoagulable state that is amenable totreatment by the antioxidant NAC amide or a derivative thereof. In anembodiment, NAC amide or a derivative thereof is useful as a wounddressing to permit enhancement of neutrophil function. In an embodiment,NAC amide or a derivative thereof is used to block the effects ofleptin, which is a cardiovascular risk factor in diabetic patients. Inan embodiment, NAC amide or a derivative thereof is used in thetreatment of total plasma homocysteine and cysteine levels withincreased urinary excretion, as well as in the treatment forhyperhomocysteinemic conditions, to improve oxidative stress. It hasbeen found that elevated levels of homocysteine pose a significant riskin vascular disease, such as atherosclerosis, venous thrombosis, heartattack and stroke, as well as neural tube defects and neoplasia.Homocysteine promotes free radical reactions. In patients with defectivehomocysteine metabolism, relatively high levels of homocysteine arepresent in the blood. Thus, in accordance with this invention, NAC amideor a derivative thereof is administered to patients with elevatedhomocysteine levels. In an embodiment, NAC amide or a derivative thereofis used as a chemoprotectant against bone marrow toxicity after orduring chemotherapy, e.g., alkylators, with or without accompanyingglutathione depletion. In an embodiment, NAC amide or a derivativethereof is used in the treatment of lithium induced renal failure. In anembodiment, NAC amide or a derivative thereof is used in the treatmentof prostatic inflammation, which may contribute to prostaticcarcinogenesis and inflammation.

In another embodiment, NAC amide or a derivative thereof is used inpulmonary disease medicine, particularly in oxygen-mediated lungdisease. NAC amide or a derivative thereof can improve oxygenation incardiopulmonary bypass during coronary artery surgery and is useful inthe treatment of chronic obstructive pulmonary disease and pulmonaryhypertension. In an embodiment, NAC amide or a derivative thereof isused in the treatment of injury in the lung due to high-energy impulsenoise-blasts, which can induce antioxidant depletion. Thus, theadministration of NAC amide or its derivatives provide an advantageousantioxidant source. NAC amide or a derivative thereof is particularlyuseful if provided as a supplement prior to noise blast exposure. NACamide or a derivative thereof is useful in the treatment of asthma withincreased oxidative stress. NAC amide or a derivative thereof is usefulfor the treatment of adult respiratory distress syndrome; in thetreatment of pulmonary fibrosis, in the treatment of idiopathicpulmonary fibrosis and asbestos exposure; and in the treatment ofchronic lung rejection. Further, NAC amide or a derivative thereof iscontemplated for use in occupational isocyanate exposure and thedevelopment of isocyanate allergy, which is believed to develop by twoprocesses, namely, isocyanate-protein conjugation and airway epithelialcell toxicity. More specifically, NAC amide or a derivative thereof canserve to protect against hexamethylene diisocyanate (HDI) conjugation tocellular proteins and to reduce HDI toxicity to human airway epithelialcells following isocyanate exposure. Thus, NAC amide or a derivativethereof can help to prevent the development of allergic sensitizationand asthma that are associated with this occupational hazard.

In another embodiment, the present invention encompasses the use of NACamide or a derivative thereof to inhibit HIV replication in chronicallyand acutely infected cells. NAC amide can be used in GSH replacementtherapy, as NAC amide and its derivatives may interfere with theexpression of the integrated HIV genome, thus, attacking the virus in amanner that is different from that of the currently employedanti-retrovirals, e.g., AZT, ddI, ddC or D4T. NAC amide or a derivativethereof can also be beneficial in countering the excess free radicalreactions in HIV infection, which may be attributable to: 1) thehypersecretion of TNF-α by B-lymphocytes in HIV infection, and 2) thecatalysis of arachidonic acid metabolism by the gp120 protein of HIV.The physiologic requirements for antioxidants by key cell types of theimmune system, and the ability of macrophages to take up intercellularantioxidants, as well as to metabolically interact with T-lymphocytes toindirectly cause their antioxidant levels to increase, offer additionalreasons that NAC amide or a derivative thereof is useful for correctingantioxidant deficiency in patients with HIV/AIDS. NAC amide and itsderivatives can serve as a suppressant of viral and bacterial species invaginal tissues by the use of intravaginal placement of gel inducedthiol.

Because HIV is known to start pathologic free radical reactions whichlead to the destruction of antioxidant molecules, as well as theexhaustion of GSH and destruction of cellular organelles andmacromolecules, NAC amide and its derivatives can be used to restoreantioxidant levels in a mammal in need thereof, to arrest thereplication of the virus at a unique point, and specifically prevent theproduction of toxic free radicals, prostaglandins, TNF-α, interleukins,and a spectrum of oxidized lipids and proteins that areimmunosuppressive and cause muscle wasting and neurological symptoms.The administration of NAC amide or a derivative thereof to elevate orreplace antioxidant levels could slow or stop the diseases progressionsafely and economically.

Because certain viral infections, such as infection by HIV, areassociated with reduced antioxidant levels, an aspect of this inventionis to increase intracellular levels of antioxidant in infected cells, aswell as to increase extracellular of antioxidant, by introducing oradministering AD3 so as to interfere with the replication of HIV and toprevent, delay, reduce or alleviate the cascade of events that areassociated with HIV infection. Because AIDS may also be associated withreduced GSSG levels, providing an amount of NAC amide to cells and/or toan individual in need thereof, can overcome any interference with denovo synthesis of antioxidant such as GSH, as well as the oxidation ofexisting GSH, which may occur in HIV infected cells. In accordance withthe present invention NAC amide or a derivative thereof is used toinhibit cytokine-stimulated HIV expression and replication in acutelyinfected cells, chronically infected cells, and in normal peripheralblood mononuclear cells. NAC amide or derivatives thereof can be used toeffect concentration-dependent inhibition of HIV expression induced byTNF-α or IL-6 in chronically infected cells. Due to NAC amide's superiorability to cross cellular membranes and enhanced lipophilic properties,NAC amide and derivatives thereof can be used at lower concentrations ascompared to NAC or GSH, such as 2-fold, 5-fold, 10-fold, 100-fold,1000-fold, 10,000-fold or lower, concentrations.

Further, the depletion of antioxidants by HIV in infected cells is alsoassociated with a process known as apoptosis, or programmed cell death.By providing NAC amide or a derivative thereof to HIV infectedindividuals and/or cells, the intercellular processes, whichartificially deplete GSH and which may lead to cell death can beprevented, interrupted, or reduced. Similarly, the NAC amide thiol canbe used as a blocker of bio-replication from West Nile Virus andprotection of cells from the cytopathic effect after infection of WestNile Virus, as well as other RNA and DNA virus infections.

In accordance with the invention, NAC amide or a derivative thereof maybe administered by several routes that are suited to the treatment ortherapy method, as will be appreciated by the skilled practitioner.Nonlimiting examples of routes and modes of administration for NAC amideand its derivatives include parenteral routes of injection, includingsubcutaneous, intravenous, intramuscular, and intrasternal. Other modesof administration include, but are not limited to, oral, inhalation,topical, intranasal, intrathecal, intracutaneous, opthalmic, vaginal,rectal, percutaneous, enteral, injection cannula, timed release andsublingual routes. Administration of NAC amide and its derivatives mayalso be achieved through continuous infusion. In one embodiment of thepresent invention, administration of NAC amide and its derivatives maybe mediated by endoscopic surgery. For the treatment of variousneurological diseases or disorders that affect the brain, NAC amide or aderivative thereof can be introduced into the tissues lining theventricles of the brain. The ventricular system of nearly all brainregions permits easier access to different areas of the brain that areaffected by the disease or disorder. For example, for treatment, adevice, such as a cannula and osmotic pump, can be implanted so as toadminister a therapeutic compound, such as NAC amide, or derivativethereof as a component of a pharmaceutically acceptable composition.Direct injection of NAC amide and its derivatives are also encompassed.For example, the close proximity of the ventricles to many brain regionsis conducive to the diffusion of a secreted or introduced neurologicalsubstance in and around the site of treatment by NAC amide.

For administration to a recipient, for example, injectableadministration, a composition or preparation formulated to containwater-soluble NAC amide or a derivative thereof is typically in asterile solution or suspension. Alternatively, NAC amide or a derivativethereof can be resuspended in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Non-limiting examples of excipients suitable for use includewater, phosphate buffered saline (pH 7.4), 0.15M aqueous sodium chloridesolution, dextrose, glycerol, dilute ethanol, and the like, and mixturesthereof. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids, which maybe used either on their own or as admixtures.

Formulations comprising NAC amide or a derivative thereof for topicaladministration may include but are not limited to lotions, ointments,gels, creams, suppositories, drops, liquids, sprays and powders. NACamide or a derivative thereof may be administered to mucous membranes inthe form of a liquid, gel, cream, and jelly, absorbed into a pad orsponge. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.Compositions comprising NAC amide or a derivative thereof for oraladministration include powders or granules, suspensions or solutions inwater or non-aqueous media, sachets, capsules or tablets. Thickeners,diluents, flavorings, dispersing aids, emulsifiers or binders may bedesirable. Formulations for parenteral administration may include, butare not limited to, sterile solutions, which may also contain buffers,diluents and other suitable additives.

The present invention also provides a food additive comprising NAC amideor a derivative thereof for mammalian, preferably human, consumption.NAC amide and other cysteine derivatives have been detected in manydifferent food products, including but not limited to, garlic, peppers,turmeric, asparagus, and onions. See, for example, Hsu, C. C., et al,(2004) J. Nutr. 134:149-152 and Demirkol, O. et al, (2004) J. Agric.Food Chem. 52. The food additive can comprise NAC amide or itsderivative in a liquid or solid material intended to be added to afoodstuff. The food additives can be added to “food compositions”including any products—raw, prepared or processed—which are intended forhuman consumption in particular by eating or drinking and which maycontain nutrients or stimulants in the form of minerals, carbohydrates(including sugars), proteins and/or fats, and which have been modifiedby the incorporation of a food additive comprising NAC amide or aderivative of NAC amide as provided herein. The present modified foodcompositions can also be characterized as “functional foodstuffs or foodcompositions”. “Foodstuffs” can also be understood to mean pure drinkingwater.

The term “food additive” is understood to mean any a liquid or solidmaterial intended to be added to a foodstuff. This material can, forexample, have a distinct taste and/or flavor, such as a salt or anyother taste or flavor potentiator or modifier. It is to be noted,however, that the food additive comprising NAC amide or a NAC amidederivative does not necessarily have to be an agent having a distincttaste and/or flavor.

Other food additives that can be added in combination with NAC amide, orin food additive formulations of NAC amide include, but are not limitedto, acids which are added to make flavours “sharper”, and also act aspreservatives and antioxidants, such as vinegar, citric acid, tartaricacid, malic acid, fumaric acid, lactic acid, acidity regulators,anti-caking agents, antifoaming agents, antioxidants such as vitamin Cand tocopherols such as vitamin E, bulking agents, such as starch areadditives, food coloring, color retention agents, emulsifiers flavors,flavor enhancers, humectants, preservatives, propellants, stabilizers,thickeners and gelling agents, like agar or pectin, and sweeteners .

Doses, amounts or quantities of NAC amide, or derivative thereof as wellas the routes of administration used, are determined on an individualbasis, and correspond to the amounts used in similar types ofapplications or indications known to those having skill in the art. Asis appreciated by the skilled practitioner in the art, dosing isdependent on the severity and responsiveness of the condition to betreated, but will normally be one or more doses per day, with course oftreatment lasting from several days to several months, or until a cureis effected or a diminution of disease state is achieved. Personsordinarily skilled in the art can easily determine optimum dosages,dosing methodologies and repetition rates. For example, a pharmaceuticalformulation for orally administrable dosage form can comprise NAC amide,or a pharmaceutically acceptable salt, ester, or derivative thereof inan amount equivalent to at least 25-500 mg per dose, or in an amountequivalent to at least 50-350 mg per dose, or in an amount equivalent toat least 50-150 mg per dose, or in an amount equivalent to at least25-250 mg per dose, or in an amount equivalent to at least 50 mg perdose. NAC amide or a derivative thereof can be administered to bothhuman and non-human mammals. It therefore has application in both humanand veterinary medicine.

Examples of suitable esters of NAC amide include alkyl and aryl esters,selected from the group consisting of methyl ester, ethyl ester,hydroxyethyl ester, t-butyl ester, cholesteryl ester, isopropyl esterand glyceryl ester.

As described herein, a number of conditions, diseases and pathologiesare believed to be associated with reduced intracellular antioxidantlevels, including AIDS, diabetes, macular degeneration, congestive heartfailure, cardiovascular disease and coronary artery restenosis, lungdisease, asthma, virus infections, e.g., toxic and infectious hepatitis,rabies, HIV; sepsis, osteoporosis, toxin exposure, radiation exposure,burn trauma, prion disease, neurological diseases, blood diseases,arterial disease, muscle disease, tumors and cancers. Many of thesediseases and conditions may be due to insufficient glutathione levels.Further, exposure to toxins, radiation, medications, etc., may result infree radical reactions, including types of cancer chemotherapy.Accordingly, the present invention provides NAC amide or a derivativethereof as an agent that can treat these diseases and conditions in aconvenient and effective formulation, particularly for oraladministration. The administration of exogenous NAC amide or aderivative thereof can serve to supplement or replace the hepatic outputof GSH and to assist in the maintenance of reduced conditions within theorganism. The failure to alleviate free radical reactions allows anundesirable cascade that can cause serious damage to macromolecules, aswell as lipid peroxidation and the generation of toxic compounds.Maintaining adequate levels of GSH is necessary to block these freeradical reactions. When natural GSH levels are debilitated orjeopardized, NAC amide or a derivative thereof is able to provideefficient and effective remedial action.

NAC amide can form chelation complexes with copper and lead. NAC amidemay also form circulating complexes with copper in the plasma. Thus, NACamide or a derivative thereof can be administered to treat metaltoxicity. NAC amide-metal complexes will be excreted, thus reducing themetal load. Thus, NAC amide or a derivative thereof may be administeredfor the treatment of toxicity associated with various metals, e.g.,iron, copper, nickel, lead, cadmium, mercury, vanadium, manganese,cobalt, transuranic metals, such as plutonium, uranium, polonium, andthe like. It is noted that the chelation properties of NAC amide areindependent from its antioxidant properties. However, because some metaltoxicities are free radical mediated, e.g., iron, NAC amideadministration may be particularly advantageous for such conditions.

In order to provide high bioavailability, NAC amide or a derivativethereof can be provided in a relatively high concentration in proximityto the mucous membrane, e.g., the duodenum for oral administration.Thus, NAC amide or a derivative thereof can be administered as a singlebolus on an empty stomach. The preferred dosage is between about100-10,000 mg NAC amide or between about 250-3,000 mg NAC amide.Further, the NAC amide or NAC amide derivative formulation can bestabilized with a reducing agent, e.g., ascorbic acid, to reduceoxidation both during storage and in the digestive tract prior toabsorption. The use of crystalline ascorbic acid has the added benefitof providing improved encapsulation and serving as a lubricant for theencapsulation apparatus. Capsules, e.g., a two-part gelatin capsule, aredosage forms that protect NAC amide from air and moisture, whiledissolving quickly in the stomach. The capsule is preferably a standardtwo-part hard gelatin capsule of double-O (OO) size, which may beobtained from a number of sources. After filling, the capsules arepreferably stored under nitrogen to reduce oxidation during storage. Thecapsules are preferably filled according to the method of U.S. Pat. No.5,204,114, incorporated herein by reference in its entirety, usingcrystalline ascorbic acid as both an antistatic agent and stabilizer.Further, each capsule preferably contains 500 mg of NAC amide and 250 mgof crystalline ascorbic acid. A preferred composition includes no otherexcipients or fillers; however, other compatible fillers or excipientsmay be added. While differing amounts and ratios of NAC amide andstabilizer may be used, these amounts are preferable because they fill astandard double-O capsule, and provide an effective stabilization andhigh dose. Further, the addition of calcium carbonate is avoided as itmay contain impurities and may accelerate the degradation of NAC amidein the small intestine due to its action as a base, which neutralizesstomach acid.

NAC amide or a derivative thereof is advantageously administered overextended periods. Therefore, useful combinations include NAC amide orNAC amide derivatives and drugs intended to treat chronic conditions.Such drugs are well absorbed on an empty stomach and do not have adverseinteractions or reduced or variable combined absorption. One particularclass of drugs includes central or peripheral adrenergic orcatecholenergic agonists, or reuptake blockers, which may produce anumber of toxic effects, including neurotoxicity, cardiomyopathy andother organ damage. These drugs are used, for example, as cardiac,circulatory and pulmonary medications, anesthetics and psychotropic/antipsychotic agents. Some of these drugs also have abuse potential, asstimulants, hallucinogens, and other types of psychomimetics. Other freeradical initiation associated drugs include thorazine, tricyclicantidepressants, quinolone antibiotics, benzodiazepines, acetaminophenand alcohol. Accordingly, NAC amide or a derivative thereof canadvantageously be provided in an oral pharmaceutical formulation in anamount of between about 50-10,000 mg, along with an effective amount ofa pharmacological agent that is capable of initiating free radicalreactions in a mammal. The pharmacological agent is, for example, anadrenergic, dopaminergic, serotonergic, histaminergic, cholinergic,gabaergic, psychomimetic, quinone, quinolone, tricyclic, and/or steroidagent.

In the following aspects of the invention, formulations of NAC amide ora derivative thereof provide an advantageous alternative to GSHadministration. NAC amide or a derivative thereof offers beneficialproperties of lipophilicity and cell-permeability, allowing it to morereadily enter cells and infiltrate the blood-brain barrier more readilythan GSH, NAC or other compounds. The properties of NAC amide or aderivative thereof may increase its bioavailability followingadministration to provide an improved treatment for the variousdiseases, disorders, pathologies and conditions as described herein.

Hepatic glutathione is consumed in the metabolism, catabolism and/orexcretion of a number of agents, including aminoglycoside antibiotics,acetominophen, morphine and other opiates. The depletion of hepaticglutathione may result in hepatic damage or a toxic hepatitis. High doseniacin, used to treat hypercholesterolemia, has also been associatedwith a toxic hepatitis. The present invention therefore encompasses anoral pharmaceutical formulation comprising NAC amide or a derivativethereof in an amount between about 50-10,000 mg, administered inconjunction with an effective amount of a pharmacological agent thatconsumes hepatic glutathione reserves.

A number of pathological conditions result in hepatic damage. Thisdamage, in turn, reduces the hepatic reserves of glutathione and theability of the liver to convert oxidized glutathione to its reducedform. Other pathological conditions are associated with impairedglutathione metabolism. These conditions include both infectious andtoxic hepatitis, cirrhosis, hepatic primary and metastatic carcinomas,traumatic and iatrogenic hepatic damage or resection. The presentinvention encompasses a pharmaceutical formulation comprising NAC amideor a derivative of NAC amide and an antiviral or antineoplastic agent.The antiviral or antineoplastic agent is, for example, a nucleosideanalog.

Glutathione is degraded, and cysteine is excreted, possibly in theurine. Very high doses of glutathione may therefore result incysteinuria, which may result in cysteine stones. Other long termtoxicity or adverse actions may result. Therefore, a daily intake ofgreater than about 10 gm for extended period should be medicallymonitored. On the other hand, individual doses below about 50 mg areinsufficient to raise the concentration of the duodenal lumen to highlevels to produce high levels of absorption, and to provide clinicalbenefit. Therefore, the formulations according to the present inventionhave an NAC amide or NAC amide derivative content greater than 50 mg,and are provided in one or more doses totaling up to about 10,000 mg perday.

In the treatment of HIV infection, it is believed that the oraladministration of a relatively high dose bolus of glutathione, i.e., 1-3grams per day, on an empty stomach, will have two beneficial effects.First, HIV infection is associated with a reduction in intracellularglutathione levels in PBMs, lung, and other tissues. It is furtherbelieved that by increasing the intracellular glutathione levels, thefunctioning of these cells may be returned to normal. Therefore, theadministration of NAC amide or a derivative thereof according to thepresent invention will treat the effects of HIV infection. Oraladministration of NAC amide, or derivative thereof, optionally incombination with ascorbic acid and/or with an antiretroviral agent. Itis noted that the transcription mechanisms and control involved inretroviral infection is believed to be relatively conserved among thedifferent virus types. Therefore, late stage retroviral suppression isexpected for the various types of human retroviruses and analogousanimal retroviruses. It has also been found in in vitro tests that byincreasing the intracellular levels of glutathione in infected monocytesto the high end of the normal range, the production of HIV from thesecells may be suppressed for about 35 days. This is believed to berelated to the interference in activation of cellular transcription ofcytokines, including NF-κB and TNF-α. Therefore, the infectivity of HIVinfected persons may be reduced, helping to prevent transmission. Thisreduction in viral load may also allow the continued existence ofuninfected but susceptible cells in the body.

NAC amide, or derivative thereof administered according to the presentmethod, can be use in the treatment of congestive heart failure (CHF).In CHF, there are believed to be two defects. First, the heart muscle isweakened, causing enlargement of the heart. Second, peripheral vasospasmis believed to be present, causing increased peripheral resistance. NACamide or a derivative thereof can be effective in enhancing the effectsof nitric oxide, and therefore can be of benefit to these patients bydecreasing vasoconstriction and peripheral vascular resistance, whileincreasing blood flow to the tissues. The present invention thusencompasses the oral administration of NAC amide or a derivative thereofin conjunction with a congestive heart failure medication, for example,digitalis glycosides, dopamine, methyldopa, phenoxybenzamine,dobutamine, terbutaline, aminone, isoproterenol, beta blockers, calciumchannel blockers, such as verapamil, propranolol, nadolol, timolol,pindolol, alprenolol, oxprenolol, sotalol, metoprolol, atenolol,acebutolol, bevantolol, tolamolol, labetalol, diltiazem, dipyridamole,bretylium, phenyloin, quinidine, clondine, procainamide, acecamide,amiodarione, disopyramide, encamide, flecanide, lorcamide, mexiletine,tocamide, captopril, minoxodil, nifedipine, albuterol, pargyline,vasodilators, including nitroprusside, nitroglycerin, phentolamine,phenoxybeizamine, hydrazaline, prazosin, trimazosin, tolazoline,trimazosin, isosorbide dinitrate, erythrityl tetranitrate, aspirin,papaverine, cyclandelate, isoxsuprine, niacin, nicotinyl alcohol,nylidrin, diuretics, including furosemide, ethacrynic acid,spironolactone, triamterine, amiloride, thiazides, bumetanide, caffeine,theophylline, nicotine, captopril, salalasin, and potassium salts.

In another of its embodiments, the present invention embraces NAC amideor a derivative thereof to treat hepatitis of various types by oraladministration. For example, both alcohol and acetaminophen arehepatotoxic and result in reduced hepatocyte glutathione levels.Therefore, these toxicities may be treated according to the presentinvention with the use of NAC amide or a derivative thereof. NAC amideand its derivatives may also be effective in the treatment of toxicitiesto other types of cells or organs, which result in free radical damageto cells or reduced glutathione levels.

Diabetes, especially uncontrolled diabetes, results in glycosylation ofvarious enzymes and proteins, which may impair their function orcontrol. In particular, the enzymes which produce reduced glutathione(e.g., glutathione reductase) become glycosylated and non-functional.Therefore, diabetes is associated with reduced glutathione levels, andin fact, many of the secondary symptoms of diabetes may be attributed toglutathione metabolism defects. According to this invention, NAC amideor a derivative thereof can be used to supplement diabetic patients inorder to prevent a major secondary pathology. The present invention alsoencompasses an oral pharmaceutical formulation comprising NAC amide andan antihyperglycemic agent.

High normal levels of glutathione deactivate opiate receptors. Thus, theadministration of NAC amide or a derivative thereof may be of benefitfor treating obesity and/or eating disorders, other addictive orcompulsive disorders, including tobacco (nicotine) and opiate additions.This invention also encompasses administering NAC amide or a derivativethereof in conjunction with nicotine. The physiologic effects ofnicotine are well known. NAC amide or a derivative thereof may causevasodilation and improve cerebral blood flow, thereby resulting in asynergistic cerebral function-enhancing effect.

In mammals, the levels of glutathione in the plasma are relatively low,in the micromolar range, while intracellular levels are typically in themillimolar range. Therefore, intracellular cytosol proteins aresubjected to vastly higher concentrations of glutathione thanextracellular proteins. The endoplasmic reticulum, a cellular organelle,is involved in processing proteins for export from the cell. It has beenfound that the endoplasmic reticulum forms a separate cellularcompartment from the cytosol, having a relatively oxidized state ascompared to the cytosol, and thereby promoting the formation ofdisulfide links in proteins, which are often necessary for normalactivity. In a number of pathological states, cells may be induced toproduce proteins for export from the cells, and the progression of thepathology is interrupted by interference with the production and exportof these proteins. For example, many viral infections rely on cellularproduction of viral proteins for infectivity. The interruption of theproduction of these proteins will interfere with infectivity. Likewise,certain conditions involve specific cell-surface receptors, which mustbe present and functional. In both cases, cells that are induced toproduce these proteins will deplete reduced glutathione in theendoplasmic reticulum. It is noted that cells that consume glutathionewill tend to absorb glutathione from the plasma, and may be limited bythe amounts present. Therefore, by increasing plasma glutathione levels,even transiently, the reducing conditions in the endoplasmic reticulummay be interfered with, and the protein production blocked. Normal cellsmay also be subjected to some interference; however, in viral infectedcells, or cells otherwise abnormally stimulated, the normal regulatorymechanisms may not be intact, and the redox conditions in theendoplasmic reticulum will not be controlled by the availability ofextracellular glutathione. The administration of NAC amide or itsderivatives may serve to replenish GSH or the effects of GSH and providesignificant effects for such conditions.

Reproduction of herpes viruses, which are DNA viruses, is inhibited orreduced in cell culture by the administration of extracellularglutathione. Examples of DNA viruses include Herpes Simplex Virus I,Herpes Simplex Virus II, Herpes zoster, cytomegalovirus, Epstein Barrvirus and others. Therefore, according to the present invention, DNAvirus and herpes virus infections may be treated by administering NACamide or a derivative thereof. In addition, infection by the rabiesvirus, an RNA virus, may be treated by the administration ofglutathione. While standard treatments are available, and indeedeffective when timely administered, glutathione may be useful in certaincircumstances. Therefore, rabies virus infection may be treated, atleast in part, by administering NAC amide or a derivative thereofaccording to the present invention. One available treatment for rabiesis an immune serum. The present invention encompasses the parenteraladministration of NAC amide, or derivative thereof separately, or incombination with one or more immunoglobulins.

Coronary heart disease risk is increased by the consumption of ahigh-fat diet and is reduced by the intake of antioxidant vitamins,including vitamin E and vitamin C, as well as flavonoids. High fat mealsimpair the endothelial function through oxidative stress, resulting inimpaired nitric oxide availability. It has been found that vitamin C andvitamin E restore the vasoconstriction resulting from nitric oxideproduction by endothelium after a high fat meal. According to thepresent invention, NAC amide or a derivative thereof may be administeredprophylactically to combat vascular disease.

There are known to be qualitative differences among several species offree radicals. Accordingly, their rates of formation will differ, aswill the different types of inciting agents that may have to besimultaneously controlled. For example, for those with maculardegeneration, continued, unprotected exposure of the eyes to strongsunlight and to tobacco smoke would limit the benefits from anantioxidant used as a therapeutic agent for control of this disease.Therefore, one aspect of the invention provides synergistic therapies topatients by increasing antioxidant levels systemically or in specificorgans as well as reducing oxidative, free radical generating andionizing influences. In this case, NAC amide therapy would becomplemented with ultraviolet blocking sunglasses, and a tobacco smokingcessation plan, as necessary. NAC amide or a derivative thereof can beused in combination with alpha tocopherol succinate, if necessary. Freeradicals occur in different parts or subparts of tissues and cells, withdifferent inciting agents. For example, in trauma to the brain or spinalcord, the injurious free radicals are in the fatty (lipid) coveringsthat insulate nerve fibers, i.e., the myelin sheaths. Extremely highdoses of a synthetic corticosteroid, 5 to 10 grams of methylprednisolone sodium succinate (MPSS), given for just 24 hours, rapidlyreach the brain and spinal cord and diffuse rapidly into the myelin,neutralizing the trauma-induced radicals. The present inventiontherefore provides a pharmaceutical composition comprising a combinationof NAC amide or a NAC amide derivative and a glucocorticoid agent.

According to the present invention, orally administered NAC amide or aderivative thereof can raise cell levels of glutathione to inhibit anumber of pathologic processes. For example, NAC amide can be used tocurtail the virtually self-perpetuating, powerful biochemical cyclesproducing corrosive free radicals and toxic cytokines that are largelyresponsible for the signs and symptoms of AIDS. These biochemical cyclesdestroy considerable quantities of glutathione but they can eventuallybe brought under control, and normalized with sufficient, ongoing NACamide therapy. A typical example is the over production of a substance,15 HPETE (15-hydroperoxy eicosatetraenoic acid), from activatedmacrophages. 15 HPETE is a destructive, immunosuppressing substance andrequires glutathione for conversion into a non-destructive, benignmolecule. The problem is that once macrophages are activated, they aredifficult to normalize. Once inside cells, GSH curtails the productionof free radicals and cytokines, corrects the dysfunctions of lymphoctyesand of macrophages, reinforces defender cells in the lungs and otherorgans and halts HIV replication in all major infected cell types, bypreventing the activation of the viral DNA by precluding the activationof NF-κB, inhibiting the TAT gene product of HIV that drives viralreplication and dismantling the gp120 proteins of the virus coat. NACamide can be provided to disrupt the gp120 protein, thereby offering apotential mode of preventing transmission of virus not only to othercells in the patient, but perhaps to others.

Besides classic antiviral or antiretroviral agents (reversetranscriptase inhibitors, protease inhibitors), a number of othertherapies may be of benefit for AIDS patients, and the present inventionprovides combinations of NAC amide or a derivative thereof with thefollowing drugs: cycloporin A, thalidomide, pentoxifylline, selenium,desferroxamine, 2L-oxothiazolidine, 2L-oxothiazolidine-4-carboxylate,diethyldithiocarbamate (DDTC), BHA, nordihydroguairetic acid (NDGA),glucarate, EDTA, R-PIA, alpha-lipoic acid, quercetin, tannic acid,2′-hydroxychalcone, 2-hydroxychalcone, flavones, alpha-angelicalactone,fraxetin, curcurmin, probucol, and arcanut (areca catechul).

Inflammatory responses are accompanied by large oxidative bursts,resulting in large numbers of free radicals. Therefore, NAC amide andits derivatives may have application in the therapy for inflammatorydiseases. NAC amide or a derivative thereof may advantageously reducethe primary insult, as well as undesired aspects of the secondaryresponse. According to the present invention, NAC amide or a derivativethereof may be administered to patients suffering from an inflammatorydisease, such as arthritis of various types, inflammatory bowel disease,etc. The present invention also provides combination pharmaceuticaltherapy including NAC amide or NAC amide derivative and an analgesic oranti-inflammatory agent, for example, opiate agonists, glucocorticoidsor non-steroidal anti-inflammatory drugs (NSAIDS), including opiumnarcotics, meperidine, propoxyphene, nalbuphine, pentazocine,buprenorphine, aspirin, indomethacin, diflunisal, acetominophen,ibuprofen, naproxen, fenoprofen, piroxicam, sulindac, tolmetin,meclofenamate, zomepirac, penicillamine, phenylbutazone,oxyphenbutazone, chloroquine, hydroxychloroquine, azathiaprine,cyclophosphamide, levamisole, prednisone, prednisolone, betamethasone,triamcinolone, and methylprednisolone. NAC amide and its derivatives mayalso be beneficial for the treatment of parotitis, cervical dysplasia,Alzheimer's disease, Parkinson's disease, aminoquinoline toxicity,gentamycin toxicity, puromycin toxicity, aminoglycoside nephrotoxicity,paracetamol, acetaminophen and phenacetin toxicity.

NAC amide or a derivative thereof may be added to a virus-contaminatedfluid or potentially contaminated fluid to inactivate the virus. Thisoccurs, for example, by reduction of critical viral proteins. Accordingto an embodiment, NAC amide or a derivative thereof is added to blood orblood components prior to transfusion. The added NAC amide or derivativeof NAC amide is added in a concentration of between about 100 micromolarto about 500 millimolar or to a solubility limit, whichever is lower,and more preferably in a concentration of about 10-50 millimolar.Additionally, the addition of NAC amide or a derivative thereof to wholeblood, packed red blood cells, or other formed blood components (whiteblood cells, platelets) may be used to increase the shelf like and/orquality of the cells or formed components.

In another embodiment, the present invention encompasses the use of NACamide, or derivative thereof or a pharmaceutically acceptable salt orester thereof, in the treatment and/or prevention of cosmetic conditionsand dermatological disorders of the skin, hair, nails, and mucosalsurfaces when applied topically. In accordance with the invention,compositions for topical administration are provided that include (a)NAC amide, or derivative thereof or a suitable salt or ester thereof, ora physiologically acceptable composition containing NAC amide; and (b) atopically acceptable vehicle or carrier. The present invention alsoprovides a method for the treatment and/or prevention of cosmeticconditions and/or dermatological disorders that entails topicaladministration of NAC amide- or NAC amide-derivative containingcompositions to an affected area of a patient. Such compositions andmethods are useful in anti-aging treatments and therapies, as well asfor the treatment of wrinkles, facial lines and depressions,particularly around the eyes and mouth, creases in the skin, age spotsand discolorations, and the like.

In another embodiment, the present invention provides methods andcompositions useful for cancer and pre-cancer therapy utilizing NACamide, or derivative thereof or its pharmaceutically acceptable salts oresters. The present invention particularly relates to methods andcompositions comprising NAC amide or a derivative thereof in whichapoptosis is selectively induced in cells of cancers or precancers. Inanother embodiment, the present invention relates to a method ofselectively inducing apoptosis of precancer cells by administering aneffective amount of NAC amide or a derivative thereof to a subject. Inthis embodiment, NAC amide or a derivative thereof can be topicallyadministered to the subject. In another embodiment, the presentinvention relates to a method of selectively inducing apoptosis incancer cells by administering an effective amount of NAC amide or aderivative thereof to a subject. NAC amide or its derivative can betopically administered to the subject in this embodiment. Selectiveapoptosis refers to a situation in which corresponding normal,non-transformed cells do not undergo NAC amide-induced cell death. Inyet another embodiment, the present invention relates to a methodcomprising reducing the number of cancer cells present in a subject byadministering NAC amide or a derivative thereof to the subject as anadjunct to chemotherapy or radiation therapies such that thesusceptibility of the cancer cells to apoptosis is enhanced relative tothe non-cancer cells of the subject. In a further embodiment, thepresent invention relates to a method comprising administering aneffective amount of NAC amide or a derivative thereof as an adjunct top53 therapy, including p53 gene therapy. The cancer or precancer cellsin which apoptosis is induced are generally those which exhibit at leastone functional p53 allele. In certain instances, administration of NACamide results in restoration of mutant p53 protein conformation and/oractivity to a functional state. It is to be understood that anendogenous functional p53 allele is not necessary for methods comprisingp53 therapy, including p53 gene therapy.

In another embodiment of the invention, methods are provided whichcomprise administering NAC amide or a derivative thereof to selectivelyinduce cells which arise in hyperproliferative or benigndysproliferative disorders. Another embodiment of the present inventionencompasses the use of NAC amide or a derivative thereof in methods forselective cell cycle arrest comprising contacting the cell with anamount of NAC amide or a derivative thereof to selectively arrest cellsat a particular stage of the cell cycle. For example, administration ofNAC amide can lead to prolonged transition through G1 phase. This cellcycle arrest may be influenced by an increase in p21 expression. Themethods of the present invention can also be utilized to reduce orinhibit tumor vascularization, or to induce differentiation in cancercells.

In another of its aspects, the present invention is directed to the useof NAC amide or a derivative thereof to treat cancers and tumors thatmay be induced by faulty signals from the microenvironment that resultin loss of tissue organization in cancerous organs and loss of genomicstability in individual cancer cells. Loss of tissue structure may leadto certain cancers. Involved in this process are matrixmetalloproteinases (MMPs), which are enzymes that are important not onlyduring an organism's development and during wound healing, but also inpromoting tumorigenesis or carcinogenesis. In particular, MMPscontribute prominently to microenvironmental signals because theseproteolytic enzymes degrade structural components of the basementmembrane and extracellular matrix (ECM) and digest the contacts thatbind epithelial cells into sheets, thereby permitting the invasion oftumor cells and metastasis. MMPs can also release cell-bound inactiveprecursor forms of growth factors; degrade cell-cell and cell-ECMadhesion molecules; activate precursor zymogen forms of other MMPs; andinactivate inhibitors of MMPs and other proteases. Further, theseenzymes induce the epithelial-mesenchymal transition, or EMT, atransition of one cell state to another that causes epithelial cells todisassociate from their neighbors, break free and acquire the ability tomove through the body. While this process is essential for normaldevelopment in the embryo, in cancers, such as breast cancer, EMTprovides mobility for tumor cells and assists tumor cells in penetratingbarriers, such as wall of lymph and blood vessels, thus facilitatingmetastasis.

MMP-3 is a particular type of metalloproteinase that has been observedto induce transformation in mammary epithelial cells in culture and intransgenic mice. MMP-3 has been found to cause normal cells to expressthe Rac1b protein, an unusual form of Rho GTPase that has previouslybeen found only in cancers. Rac1b dramatically alters the cell skeleton,which facilitates the separation and movement of epithelial cells fromsurrounding cells. (D. C. Radisky et al., 2005, Nature, 436:123-127).Changes in the cell skeleton induced by Rac1b stimulate the productionof highly reactive oxygen molecules, called reactive oxygen species(ROS), which can promote cancer by leading to tissue disorganization andby damaging genomic DNA. The increased amounts of ROS induced by Rac1bactivate major genes that control the EMT, which then begins a cascadeof massive tissue disorganization and stimulates the development ofcancer by directly affecting genomic DNA, for example, causing deletionor duplication of large regions of the DNA. By altering the tissuestructure, MMPs can also activate oncogenes and comprising the integrityof the DNA in an organism's genome.

For treating cancers, e.g., breast cancer, especially those involvingthe above-described mechanisms leading to abnormal cell structure andfunction and loss of tissue integrity, NAC amide in accordance with thepresent invention can be used to block the effects of ROS. This can beachieved, for example, by administering or introducing NAC amide or aderivative thereof to cells, tissues, and/or the body of a subject inneed thereof, to affect or target molecules in the pathways leading toepithelial-mesenchymal transition. Accordingly, NAC amide or aderivative thereof can be used to inhibit MMP-3 and its functions, suchas MMP-3-induced downregulation of epithelial cytokeratins andupregulation of mesenchymal vimentin, as well as MMP3-induced cellmotility, invasion and morphological alterations. NAC amide or aderivative thereof can also be used to target ROS indirectly ordirectly, and/or the processes by which ROS activate genes that inducethe EMT.

In another embodiment, the present invention encompasses compositionsand methods comprising NAC amide or a derivative thereof for thesuppression of allograft rejection in recipients of allografts.

In another embodiment, the present invention provides a NAC amide orderivative of NAC amide in a method of supporting or nurturing thegrowth of stem cells for stem cell transplants, particularly stem cellscultured in vitro prior to introduction into a recipient animal,including humans.

In another embodiment, the present invention provides methods ofinhibiting, preventing, treating, or both preventing and treating,central nervous system (CNS) injury or disease, neurotoxicity or memorydeficit in a subject, involving the administration of a therapeuticallyeffective amount of NAC amide, or derivative thereof or apharmaceutically acceptable composition thereof. Examples of CNSinjuries or disease include traumatic brain injury (TBI), posttraumaticepilepsy (PTE), stroke, cerebral ischemia, neurodegenerative diseases ofthe brain such as Parkinson's disease, Dementia Pugilistica,Huntington's disease, Alzheimer's disease, brain injuries secondary toseizures which are induced by radiation, exposure to ionizing or ironplasma, nerve agents, cyanide, toxic concentrations of oxygen,neurotoxicity due to CNS malaria or treatment with anti-malaria agents,and other CNS traumas. In other related embodiments, the presentinvention embraces a method of treating a subject suffering from a CNSinjury or disease comprising administering to the subject a compositioncomprising a therapeutically effective amount of NAC amide or aderivative thereof. In another embodiment, the present invention relatesto a method of preventing or inhibiting a CNS injury or disease in asubject comprising administering to the subject a composition comprisinga therapeutically effective amount of NAC amide or a derivative thereof.In other embodiments, the present invention embraces a method ofpreventing, inhibiting or treating neurotoxicity or memory deficit in asubject comprising administering to the subject a composition comprisinga therapeutically effective amount of NAC amide or a derivative thereof.Where the memory deficit may be induced by electroconvulsive shocktherapy for treating diseases and disorders such as depression andschizophrenia, the composition may be administered before theelectroconvulsive shock therapy to mitigate memory loss. In relatedembodiments, the CNS injury or disease may be traumatic brain injury(TBI), posttraumatic epilepsy (PTE), stroke, cerebral ischemia, or aneurodegenerative disease. In related embodiments, CNS injury may beinduced by fluid percussion, by trauma imparted by a blunt object, forexample on the head of the subject, by trauma imparted by an objectwhich penetrates the head of the subject, by exposure to radiation,ionizing or iron plasma, a nerve agent, cyanide, toxic concentrations ofoxygen, CNS malaria, or an anti-malaria agent. In the embodiments of thepresent invention, the therapeutically effective amount of NAC amide ora derivative thereof administered to the subject is the amount requiredto obtain the appropriate therapeutic effect, for example, about 0.001mg to about 20 mg per kg of the subject, preferably about 1 mg to about10 mg per kg of the subject, more preferably about 3 mg to about 10 mgper kg of the subject. In additional embodiments, the total daily amountof NAC amide or a derivative thereof administered to the subject isabout 50 mg to about 1200 mg, or about 100 mg to about 1000 mg, or about200 mg to about 800 mg, or about 300 mg to about 600 mg.

In other embodiments, the invention encompasses a method of treating asubject (e.g., an animal, including humans) before the subject isexposed or likely to be exposed to a risk of CNS injury or damage, orbefore the subject is exposed to conditions likely to causeneurotoxicity or memory deficit or both, by administering NAC amide or aderivative thereof to a subject in a period of time prior to theexposure of the subject to the risk of CNS injury or damage, etc.Illustratively, conditions that may cause CNS injury or damage,neurotoxicity or memory deficit include electroconvulsive shock therapy,traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke,cerebral ischemia, neurodegenerative diseases, fluid percussion, a bluntobject impacting the head of the subject, an object penetrating the headof the subject, radiation, ionizing or iron plasma, nerve agents,cyanide, toxic concentrations of oxygen, CNS malaria, and anti-malariaagents. Other conditions that may cause CNS injury or damage,neurotoxicity or memory deficit include, without limitation, certainmedical procedures or conditions associated with risk for CNS ischemia,hypoxia or embolism such as brain tumor, brain surgery, otherbrain-related disorders, open heart surgery, carotid endarterectomy,repair of aortic aneurysm, atrial fibrillation, cardiac arrest, cardiacor other catheterization, phlebitis, thrombosis, prolonged bed rest,prolonged stasis (such as during space travel or long trips viaairplane, rail, car or other transportation), CNS injury secondary toair/gas embolism or decompression sickness. The period of time may beabout 72 hours prior to the time of expected exposure, or about 48 hoursprior to the time of expected exposure, or about 12 hours prior to thetime of expected exposure, or about 4 hours prior to the time ofexpected exposure, or about 30 minutes-2 hours prior to the time ofexpected exposure. The administration of NAC amide may be continuousfrom the initial time of treatment to the end of treatment. For example,a transdermal patch or a slow-release formulation may be used tocontinually administer NAC amide or a derivative thereof to the subjectfor a given period of time. Alternatively, NAC amide or a derivativethereof may be administered to the subject periodically. For example,NAC amide or a derivative thereof may first be administered at about 24hours before the time of expected exposure and then administered atabout every 2 hours thereafter. For these embodiments of the invention,the NAC amide- or NAC amide derivative-containing composition mayfurther comprise a pharmaceutically acceptable excipient and thecomposition may be administered intravenously, intradermally,subcutaneously, orally, transdermally, transmucosally or rectally.

In other embodiments, the present invention encompasses a pharmaceuticalcomposition for treating or preventing CNS injury, disease orneurotoxicity in a subject comprising a therapeutically effective amountof NAC amide or a derivative thereof and a pharmaceutically acceptableexcipient. In a further embodiment, the invention embraces a kitcomprising a composition comprising a therapeutically effective amountof NAC amide or a derivative thereof. The kit may further comprise adevice for administering the composition to a subject such as aninjection needle, an inhaler, a transdermal patch, as well asinstructions for use.

In another embodiment of the present invention, anti-cancer treatmentsinvolving NAC amide or a derivative thereof are designed to specificallytarget cancer and tumor cells. This embodiment is directed to the use ofnano-sized particles for the in vivo and ex vivo administration of NACamide or a derivative thereof to cancer and tumor cells. According tothis embodiment, cancer cells, which display more receptors for thevitamin folic acid (or folate) and absorb more folic acid than donormal, healthy cells, are able to be preferentially targeted. To thisend, core or shell nanogels, or nanoparticles, can be functionalizedwith folic acid or folate conjugated or linked to NAC amide or aderivative thereof without disrupting or destroying the folic acidbinding site to its cell receptor. Such functionalized nanoparticles canbe introduced into a subject, particularly a folate-deprived subject,with a cancer, e.g., epithelial cancer, in whom the cancer cells haveexcess folic acid receptors which will preferentially bind the folicacid-NAC amide (or folic acid-NAC amide derivative) nanoparticles andendocytose them. Once inside the cancer cell, NAC amide or a derivativethereof exert its therapeutic effects, for example, by inhibiting ROSand/or other target molecules that play a role in initiating, fueling,and/or maintaining cancer cells, and/or ultimately killing the cancercells.

Illustratively, PAMAM dendritic polymers <5 nm in diameter can be usedas carriers of NAC amide, as described in J. F. Kukowska-Latallo et al.,2005, Cancer Res., June 15; 65(12):5317-24, to target folic acidreceptor-expressing (overexpressing) tumor and cancer cells. Acetylateddendrimers can be conjugated to folic acid as a targeting agent and thencoupled to NAC amide or a derivative thereof and either fluorescein or6-carboxytetramethylrhodamine. Alternatively, NAC amide or a derivativethereof can be coupled to folic acid to form a conjugate and theconjugate can be coupled to the nanoparticles. These conjugates can beinjected i.v. into a tumor-bearing patient or mammal, especially thosetumors that overexpress the folic acid receptor. The folate-conjugatednanoparticles can then concentrate in the tumor and tissue followingadministration, where the delivered NAC amide or NAC derivative caninteract with ROS in the cells, and/or target other molecules to killthe cancer or tumor cells. The tumor tissue localization of thefolate-targeted polymer may be attenuated by prior i.v. injection offree folic acid.

In a similar embodiment, polymers or nanoparticles can be functionalizedto display glutathione-NAC amide or glutathione-NAC amide derivativeconjugates, which can then be used to deliver NAC amide or a derivativethereof to cancer cells which display increased numbers of glutathionereceptors on their cell surfaces. The NAC amide-glutathionenanoparticles can then be targeted to those cancer cells havingglutathione receptors and preferentially endocytosed by the cells. Inthese embodiments, the present invention provides directed delivery ofNAC amide or a derivative thereof to cells, such as cancer cells thatexpress high levels of receptors for folic acid (folate) or glutathione.In accordance with these embodiments, NAC amide (“NACA”) or a derivativethereof is coupled to a ligand for a cell surface receptor (e.g., folicacid or glutathione) to form a conjugate. This NACA-ligand conjugate iscoated or adsorbed onto readily injectable nanoparticles usingprocedures known to those skilled in the art. Accordingly, thenanoparticles containing NAC amide or a derivative thereof (“nano-NACAparticles”) may be preferentially taken up by cancer or tumor cellswhere the NAC amide will exert its desired effects.

In an embodiment, the present invention is drawn to a method of directeddelivery of NAC amide or a derivative thereof to host cells expressinghigh levels of surface receptor for a ligand, comprising: a) conjugatingacetylated dendritic nanopolymers to ligand; b) coupling the conjugatedligand of step (a) to NAC amide or a derivative thereof to form NACamide-ligand nanoparticles; and c) injecting the nanoparticles of (b)into the host. In another embodiment, the present invention is drawn toa method of directed delivery of NAC amide or a derivative thereof tohost cells expressing high levels of surface receptor for a ligand,comprising: a) coupling NAC amide or a derivative thereof to the surfacereceptor ligand to form a NAC amide-ligand conjugate; b) adsorbing theNAC amide-ligand conjugate onto nanoparticles; and c) injecting thenanoparticles of (b) into the host.

Another embodiment of the present invention provides a compound of theformula I:

wherein:

-   -   R₁ is OH, SH, or S—S-Z;    -   X is C or N;    -   Y is NH₂, OH, CH₃—C═O, or NH—CH₃;    -   R₂ is absent, H, or ═O    -   R₃ is absent or

wherein: R₄ is NH or O;

-   -   R₅ is CF₃, NH₂, or CH₃

and wherein: Z is

with the proviso that if R₁ is S—S-Z, X and X′ are the same, Y and Y′are the same, R₂ and R₆ are the same, and R₃ and R₇ are the same.

In one embodiment, R₁ is S, X is C, Y is NH—CH₃, R₂ is H, R₃ is

R₄ is O, and R₅ is CH₃. In another embodiment, R₁ is S, X is N, Y isCH₃—C═O, R₂ is H, and R₃ is absent.

The present invention also provides compounds of the formula I above,wherein R₁ is S, X is C, Y is NH₂, R₂ is

═O, R₃ is R₄ is O, and R₅ is CF₃. Compounds of the present inventionalso include compounds of formula I wherein R₁ is O, X is C, Y is NH₂,R₂ is ═O, R₃ is

R₄ is O, and R₅ is CH₃. Also provided by the present invention arecompounds of formula I wherein R₁ is S, X is C, Y is OH, R₂ is absent,R₃ is

R₄ is O, and R₅ is CH₃, or wherein R₁ is S, X is C, Y is NH₂, R₂ is ═O,R₃ is

R₄ is NH, and R₅ is NH₂. Another embodiment of the present inventionprovides compounds of formula I wherein R₁ is O, X is C, Y is OH, R₂ isabsent, R₃ is

R₄ is O, and R₅ is CH₃; or wherein R₁ is S, X is C, Y is NH₂, R₂ is ═O,R₃ is

R₄ is O, and R₅ is CH₃. In a further embodiment, the present inventionprovides compounds of formula I wherein R₁ is S—S-Z, X is C, Y is NH₂,R₂ is ═O, R₃ is

R₄ is O and R₅ is CH₃.

The compounds disclosed herein can be chiral, i.e., enantiomers, such asL- and D-isomers, or can be racemic mixtures of D- and L-isomers.Preferred compounds include, but are not limited to, the following:

In one embodiment, Compounds I through XVIII comprise NAC amide or NACamide derivatives.

In another embodiment, a process for preparing an L- or D-isomer of thecompounds of the present invention are provided, comprising adding abase to L- or D-cystine diamide dihydrochloride to produce a firstmixture, and subsequently heating the first mixture under vacuum; addinga methanolic solution to the heated first mixture; acidifying themixture with alcoholic hydrogen chloride to obtain a first residue;dissolving the first residue in a first solution comprising methanolsaturated with ammonia; adding a second solution to the dissolved firstresidue to produce a second mixture; precipitating and washing thesecond mixture; filtering and drying the second mixture to obtain asecond residue; mixing the second residue with liquid ammonia, and anethanolic solution of ammonium chloride to produce a third mixture; andfiltering and drying the third mixture, thereby preparing the L- orD-isomer compound.

The base can comprise liquid ammonia or methylamine. The second solutioncomprises water, an acetate salt, and an anhydride, wherein the acetatesalt can comprise sodium acetate or sodium trifluoroacetate, and theanhydride can comprise acetic anhydride or trifluoroacetic anhydride.Alternatively, the second solution can comprise dichloromethane,triethylamine, and 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.

In addition to liquid ammonia and an ethanolic solution of ammoniumchloride, the second residue can be further mixed with sodium metal.

In some embodiments, the process further comprises dissolving the L- orD-isomer compound in ether; adding to the dissolved L- or D-isomercompound an ethereal solution of lithium aluminum hydride, ethylacetate, and water to produce a fourth mixture; and filtering and dryingthe fourth mixture, thereby preparing the L- or D-isomer compound.

The compounds of formula II and III are prepared by mixing L- orD-cystine diamide dihydrochloride with liquid ammonia; warming themixture to remove volatiles; warming mixture in vacuo to 50° C.; addinga warm methanolic solution; filtering the solution; acidifying thefiltrate with alcoholic hydrogen chloride for obtaining a first residue,dissolving the first residue in a solution of methanol saturated withammonia; concentrating to dryness; adding water, sodium acetate andacetic anhydride; raising the temperature to 50° C.; precipitating themixture and washing the mixture with water; filtering the crude solid;drying the mixture for obtaining a second residue, mixing the secondresidue with liquid ammonia; slowly adding sodium metal; removal of thesolvent; slowly adding an ethanolic solution of ammonium chloride;filtering and separating the inorganic salt; concentrating and coolingthe filtrate to obtain a third residue; and crystallizing the thirdresidue from isopropanol.

The compounds of formula IV and V are prepared by mixing L- or D-cystinediamide dihydrochloride with methylamine; warming the mixture to removevolatiles; warming mixture in vacuo to 50° C.; adding a warm methanolicsolution; filtering the solution; acidifying the filtrate with alcoholichydrogen chloride for obtaining a first residue, dissolving the firstresidue in a solution of methanol saturated with ammonia; concentratingto dryness; adding water, sodium acetate and acetic anhydride; raisingthe temperature to 50° C.; precipitating the mixture and washing themixture with water; filtering the crude solid; drying the mixture forobtaining a second residue, mixing the second residue with liquidammonia; slowly adding sodium metal; removal of the solvent; slowlyadding an ethanolic solution of ammonium chloride; filtering andseparating the inorganic salt; concentrating and cooling the filtrate toobtain a third residue; and crystallizing the third residue fromisopropanol.

The compounds of formula VII and VIII are prepared by mixing L- orD-cystine diamide dihydrochloride with ammonia; warming the mixture toremove volatiles; warming mixture in vacuo to 50° C.; adding a warmmethanolic solution; filtering the solution; acidifying the filtratewith alcoholic hydrogen chloride for obtaining a first residue,dissolving the first residue in a solution of methanol saturated withammonia; concentrating to dryness; adding water, sodium trifluoroacetateand trifluoroacetic anhydride; raising the temperature to 50° C.;precipitating the mixture and washing the mixture with water; filteringthe crude solid; drying the mixture for obtaining a second residue,mixing the second residue with liquid ammonia; slowly adding sodiummetal; removal of the solvent; slowly adding an ethanolic solution ofammonium chloride; filtering and separating the inorganic salt;concentrating and cooling the filtrate to obtain a third residue; andcrystallizing the third residue from isopropanol.

The compounds of formula XIII and XIV are prepared by mixing L- orD-cystine diamide dihydrochloride with ammonia; warming the mixture toremove volatiles; warming mixture in vacuo to 50° C.; adding a warmmethanolic solution; filtering the solution; acidifying the filtratewith alcoholic hydrogen chloride for obtaining a first residue,dissolving the first residue in a solution of methanol saturated withammonia; concentrating to dryness; adding dichloromethane,triethylamine, and 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea;lowering the temperature to 0° C.; precipitating the mixture and washingthe mixture with water; filtering the crude solid; drying the mixturefor obtaining a second residue, mixing the second residue with liquidammonia; slowly adding sodium metal; removal of the solvent; slowlyadding an ethanolic solution of ammonium chloride; filtering andseparating the inorganic salt; concentrating and cooling the filtrate toobtain a third residue; and crystallizing the third residue fromisopropanol.

The compounds of formula XI and XII are prepared by mixing L- orD-cystine diamide dihydrochloride with liquid ammonia; warming themixture to remove volatiles; warming mixture in vacuo to 50° C.; addinga warm methanolic solution; filtering the solution; acidifying thefiltrate with alcoholic hydrogen chloride for obtaining a first residue;dissolving the first residue in a solution of methanol saturated withammonia; concentrating to dryness; adding water, sodium acetate andacetic anhydride; raising the temperature to 50° C.; precipitating themixture; washing the mixture with water; filtering the crude solid;drying the mixture for obtaining a second residue; mixing the secondresidue with liquid ammonia; slowly adding sodium metal; removal of thesolvent; slowly adding an ethanolic solution of ammonium chloride;filtering and separating the inorganic salt; concentrating and coolingthe filtrate to obtain a third residue; dissolving the third residue inether; slowly adding an ethereal solution of lithium aluminum hydride;slowly adding ethyl acetate; slowly adding water; filtering andseparating the inorganic salts; concentrating and cooling the filtrateto obtain a fourth residue; and crystallizing the fourth residue fromisopropanol.

The compounds of formula XVII and XVIII are prepared by mixing L- orD-cystine diamide dihydrochloride with liquid ammonia; warming themixture to remove volatiles; warming mixture in vacuo to 50° C.; addinga warm methanolic solution; filtering the solution; acidifying thefiltrate with alcoholic hydrogen chloride for obtaining a first residue;dissolving the first residue in a solution of methanol saturated withammonia; concentrating to dryness; adding of water, sodium acetate andacetic anhydride; raising the temperature to 50° C.; precipitation ofthe mixture; washing the mixture with water; filtering the crude solid;drying the mixture for obtaining a second residue; and crystallizing thesecond residue from isopropanol.

Another embodiment of the invention provides a process for preparing anL- or D-isomer of the compounds disclosed herein, comprising mixingS-benzyl-L- or D-cysteine methyl ester hydrochloride or O-benzyl-L- orD-serine methyl ester hydrochloride with a base to produce a firstmixture; adding ether to the first mixture; filtering and concentratingthe first mixture; repeating steps (c) and (d), to obtain a firstresidue; adding ethyl acetate and a first solution to the first residueto produce a second mixture; filtering and drying the second mixture toproduce a second residue; mixing the second residue with liquid ammonia,sodium metal, and an ethanolic solution of ammonium chloride to producea third mixture; and filtering and drying the third mixture, therebypreparing the L- or D-isomer compound.

The base can comprise liquid ammonia or methylamine. The second solutioncomprises water, an acetate salt, and an anhydride, wherein the acetatesalt can comprise sodium acetate or sodium trifluoroacetate, and theanhydride can comprise acetic anhydride or trifluoroacetic anhydride.Alternatively, the second solution can comprise dichloromethane,triethylamine, and 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.

In some embodiments, the process further comprises dissolving the L- orD-isomer compound in ether; adding to the dissolved L- or D-isomercompound an ethereal solution of lithium aluminum hydride, ethylacetate, and water to produce a fourth mixture; and filtering and dryingthe fourth mixture, thereby preparing the L- or D-isomer compound.

The compounds of formula II and III are prepared by mixing S-benzyl-L-or D-cysteine methyl ester hydrochloride with a cold methanolic solutionof ammonia; passing a stream of ammonia over the mixture; sealing theflask securely; concentrating the mixture; adding ether; filtering thesolution; concentrating the filtrate; adding ether and filtering again,to obtain a residue; suspending the residue with ethyl acetate; addingacetic anhydride to this suspension; adding water, sodium acetate andacetic anhydride; raising the temperature to 65° C.; cooling themixture; filtering the crude solid; washing with ethyl acetate; dryingthe precipitate for obtaining a second residue; mixing the secondresidue with liquid ammonia; slowly adding sodium metal; removal of thesolvent; slowly adding an ethanolic solution of ammonium chloride;filtering and separating the inorganic salt; concentrating and coolingthe filtrate to obtain a third residue; and crystallizing the thirdresidue from isopropanol.

The compounds of formula IV and V are prepared by mixing S-benzyl-L- orD-cysteine methyl ester hydrochloride with a cold methanolic solution ofmethylamine; passing a stream of methylamine over the mixture; sealingthe flask securely; concentrating the mixture; adding ether; filteringthe solution; concentrating the filtrate; adding ether and filteringagain, to obtain a residue; suspending the residue with ethyl acetate;adding acetic anhydride to this suspension; adding water, sodium acetateand acetic anhydride; raising the temperature to 65° C.; cooling themixture; filtering the crude solid; washing with ethyl acetate; dryingthe precipitate for obtaining a second residue; mixing the secondresidue with liquid ammonia; slowly adding sodium metal; removal of thesolvent; slowly adding an ethanolic solution of ammonium chloride;filtering and separating the inorganic salt; concentrating and coolingthe filtrate to obtain a third residue; and crystallizing the thirdresidue from isopropanol.

The compounds of formula VII and VIII are prepared by mixing S-benzyl-L-or D-cysteine methyl ester hydrochloride with a cold methanolic solutionof ammonia; passing a stream of methylamine over the mixture; sealingthe flask securely; concentrating the mixture; adding ether; filteringthe solution; concentrating the filtrate; adding ether and filteringagain, to obtain a residue; suspending the residue with ethyl acetate;adding trifluoroacetic anhydride to this suspension; adding water,sodium trifluoroacetate and trifluoroacetic anhydride; raising thetemperature to 65° C.; cooling the mixture; filtering the crude solid;washing with ethyl acetate; drying the precipitate for obtaining asecond residue; mixing the second residue with liquid ammonia; slowlyadding sodium metal; removal of the solvent; slowly adding an ethanolicsolution of ammonium chloride; filtering and separating the inorganicsalt; concentrating and cooling the filtrate to obtain a third residue;and crystallizing the third residue from isopropanol.

The compounds of formula IX and X are prepared by mixing O-benzyl-L- orD-serine methyl ester hydrochloride with a cold methanolic solution ofammonia; passing a stream of methylamine over the mixture; sealing theflask securely; concentrating the mixture; adding ether; filtering thesolution; concentrating the filtrate; adding ether and filtering again,to obtain a residue; suspending the residue with ethyl acetate; addingacetic anhydride to this suspension; adding water, sodium acetate andacetic anhydride; raising the temperature to 65° C.; cooling themixture; filtering the crude solid; washing with ethyl acetate; dryingthe precipitate for obtaining a second residue; mixing the secondresidue with liquid ammonia; slowly adding sodium metal; removal of thesolvent; slowly adding an ethanolic solution of ammonium chloride;filtering and separating the inorganic salt; concentrating and coolingthe filtrate to obtain a third residue; and crystallizing the thirdresidue from isopropanol.

The compounds of formula XIII and XIV are prepared by mixing S-benzyl-L-or D-cysteine methyl ester hydrochloride with a cold methanolic solutionof ammonia; passing a stream of ammonia over the mixture; sealing theflask securely; concentrating the mixture; adding ether; filtering thesolution; concentrating the filtrate; adding ether and filtering again,to obtain a residue; suspending the residue with ethyl acetate; addingacetic anhydride to this suspension; adding dichloromethane,triethylamine, and 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea;lowering the temperature to 0° C.; precipitating the mixture; washingthe mixture with water; filtering the crude solid; drying the mixturefor obtaining a second residue; mixing the second residue with liquidammonia; slowly adding sodium metal; removal of the solvent; slowlyadding an ethanolic solution of ammonium chloride; filtering andseparating the inorganic salt; concentrating and cooling the filtrate toobtain a third residue; and crystallizing the third residue fromisopropanol.

The compounds of formula XI and XII are prepared by (a) mixingS-benzyl-L- or D-cysteine methyl ester hydrochloride with a coldmethanolic solution of ammonia; passing a stream of ammonia over themixture; sealing the flask securely; concentrating the mixture; addingether; filtering the solution; concentrating the filtrate; adding etherand filtering again, to obtain a residue; suspending the residue withethyl acetate; adding acetic anhydride to this suspension; adding ofwater, sodium acetate and acetic anhydride; raising the temperature to65° C.; cooling the mixture; filtering the crude solid; washing withethyl acetate; drying the precipitate for obtaining a second residue;mixing the second residue with liquid ammonia; slowly adding sodiummetal; removal of the solvent; slowly adding an ethanolic solution ofammonium chloride; filtering and separating the inorganic salt;concentrating and cooling the filtrate to obtain a third residue;dissolving the third residue in ether; slowly adding an etherealsolution of lithium aluminum hydride; slowly adding ethyl acetate;slowly adding water; filtering and separating the inorganic salts;concentrating and cooling the filtrate to obtain a fourth residue; andcrystallizing the fourth residue from isopropanol.

The compounds of formula XV and XVI are prepared by (a) mixingO-benzyl-L- or D-serine methyl ester hydrochloride with a coldmethanolic solution of ammonia; passing a stream of ammonia over themixture; sealing the flask securely; concentrating the mixture; addingether; filtering the solution; concentrating the filtrate; adding etherand filtering again, to obtain a residue; suspending the residue withethyl acetate; adding acetic anhydride to this suspension; adding ofwater, sodium acetate and acetic anhydride; raising the temperature to65° C.; cooling the mixture; filtering the crude solid; washing withethyl acetate; drying the precipitate for obtaining a second residue;mixing the second residue with liquid ammonia; slowly adding sodiummetal; removal of the solvent; slowly adding an ethanolic solution ofammonium chloride; filtering and separating the inorganic salt;concentrating and cooling the filtrate to obtain a third residue;dissolving the third residue in ether; slowly adding an etherealsolution of lithium aluminum hydride; slowly adding ethyl acetate;slowly adding water; filtering and separating the inorganic salts;concentrating and cooling the filtrate to obtain a fourth residue; andcrystallizing the fourth residue from isopropanol.

Yet another embodiment of the invention provides a process for preparinga compound as disclosed herein, comprising mixing cystaminedihydrochloride with ammonia, water, sodium acetate, and aceticanhydride to produce a first mixture; allowing the first mixture toprecipitate; filtering and drying the first mixture to produce a firstresidue; mixing the second residue with liquid ammonia, sodium metal,and an ethanolic solution of ammonium chloride to produce a secondmixture; filtering and drying the second mixture, thereby preparing thecompound.

The compound of formula VI is prepared by mixing cystaminedihydrochloride with ammonia; adding water, sodium acetate and aceticanhydride; raising the temperature to 50° C.; precipitating the mixture;washing the mixture with water; filtering the crude solid; drying themixture for obtaining a second residue; mixing the second residue withliquid ammonia; slowly adding sodium metal; removal of the solvent;slowly adding an ethanolic solution of ammonium chloride; filtering andseparating the inorganic salt; concentrating and cooling the filtrate toobtain a third residue; and crystallizing the third residue fromisopropanol.

EXAMPLES Example 1

In this Example, NAC amide was assessed for its protective effectsagainst oxidative toxicity induced by glutamate in PC12 cells.

Materials and methods: N-(1-pyrenyl)-maleimide (NPM) was purchased fromAldrich (Milwaukee, Wis., USA). N-acetylcysteine amide was obtained fromNovia Pharmaceuticals, (Israel). High-performance liquid chromatography(HPLC)-grade solvents were purchased from Fisher Scientific (Fair Lawn,N.J.). All other chemicals were purchased from Sigma (St. Louis, Mo.,USA).

Cell culture and toxicity studies: Stock culture of PC12 cells,purchased from ATCC, were grown in 75 cm² tissue culture flasks in RPMI1640, supplemented with 10% (v/v) heat-inactivated horse serum, and 5%(v/v) fetal bovine serum, to which 1% (v/v) penicillin and streptomycinwere added. Cultures were maintained at 37° C. in a humidifiedatmosphere containing 5% CO₂. The cells were passaged twice a week.Unless specified, all of the experiments were performed using Dulbecco'smodified Eagle's medium (DMEM) as differentiation medium, supplementedwith 0.5% (v/v) fetal bovine serum, 1% (v/v) penicillin andstreptomycin. PC12 cells were plated at a density of 25×10³ cells/wellin a 24-well, collagen-coated plate for morphological assessment. Theplate was divided into five groups in triplicate: 1) control: noglutamate, no NAC amide; 2) Nerve Growth Factor (NGF) control: NGF (100ng/ml), no glutamate, no NAC amide; 3) NAC amide only: NGF (100 ng/ml),no glutamate, NAC amide (750 μM); 4) glutamate only: NGF (100 ng/ml),glutamate (10 mM), no NAC amide; and 5) Glu+NAC amide: NGF (100 ng/ml),glutamate (10 mM), NAC amide (750 μM). All wells received 100 ng/ml NGFevery other day, except Group I. After one week, cells were treated ornot (control) with 10 mM glutamate, with or without NAC amide, for 24hours. Twenty-four hours later, the cells were fixed with 0.5% (v/v)glutaraldehyde in PBS and micropictures were taken.

LDH assay: For the lactate dehydrogenase (LDH) assay, cells were platedat a density of 2.5×10⁵ cells/well in a 24 well collagen-coated cultureplate and, after 24 h; the medium was replaced with fresh DMEM mediumcontaining the desired concentration of glutamate and NAC amide. Afterthe desired incubation period, the LDH activity released was determinedusing the kit as described below. For the MTS assay, cells were platedat a density of 10⁵ cells/well in a 24 well collagen-coated plate. Atthe end of the experiments, cell viability was assayed using the kit asdescribed. The LDH activity assay was performed with the CytoTox96®Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, Wis., USA),which quantitatively measured the activity of LDH, a stable cytosolicenzyme that is released upon cell lysis [Technical Bulletin No. 163,Promega]. LDH in culture supernatants was measured with a 30-minutecoupled enzymatic assay, which resulted in the conversion of atetrazolium salt into a red formazan product. The amount of color formedwas proportional to the degree of damage to the cell membranes.Absorbance data were collected using a BMG microplate reader (BMGLabtechnologies, Inc., Durham, N.C., USA) at 490 nm. LDH leakage wasexpressed as the percentage (%) of the maximum LDH release in the cellstreated with glutamate alone (100%), according to the formula:

${\% \mspace{14mu} {LDH}\mspace{14mu} {released}} = {\frac{{Experimental}\mspace{14mu} {LDH}\mspace{14mu} {release}}{{Maximum}\mspace{14mu} {LDH}\mspace{14mu} {release}} \times 100}$

MTS assay: The MTS assay (Cell Titer 96® Aqueous One solution cellproliferation Assay, Promega) is a cell proliferation assay in which theadministered (3-(4,5-dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS) [21] isbioreduced by viable cells to a colored formazan product that is solublein media. Absorbance at 490 nm is proportional to the number of livingcells in the culture.

GSH measurement: Cellular levels of GSH were determined by using themethod as described in Winters R. A. et al., Anal Biochem.,227(1):14-21, 1995. Cells were seeded at a density of 80,000 cells/cm²on poly-D-lysine coated (0.05 mg/ml) 75 cm² flasks (5 ml/flask) for GSHmeasurement. After 24 hours, the flasks were incubated with fresh mediumcontaining glutamate (10 mM), or BSO (0.2 mM) or Glu+BSO+NAC amide (750μM) at 370 C for another 24 h. After the incubation period, cells wereremoved from the cultures and homogenized in serine borate buffer (100mM Tris-HCl, 10 mM boric acid, 5 mM L-serine, 1 mM DETAPAC, pH 7.4).Twenty (20) μl of the diluted cell homogenate were added to 2301 ofserine borate buffer and 750 μl of NPM (1 mM in acetonitrile). Theresulting solutions were incubated at room temperature for 5 min. Thereaction was stopped by the addition of 5 μl of 2N HCl. The samples werethen filtered through a 0.2 μm Acrodisc filter and injected onto theHPLC system.

MDA measurement: To prepare the solution, 350 μl of straight cellhomogenate, 100 μl of 500 ppm BHT (butylated hydroxytoluene), and 550 μlof 10% TCA (trichloroacetic acid) were combined and boiled for 30 min.The tubes were cooled on ice and centrifuged for 10 min at 2500 rpm.Five hundred (500) μl of the supernatant were removed and 5001 of TBA(thiobarbituric acid) were added. The tubes were boiled again for 30min, and then cooled on ice. From this solution, 500 μl were removed,added to 1.0 ml of n-butanol, vortexed, and centrifuged for 5 min at 60g to facilitate a phase separation. The top layer was then filteredthrough 0.45 μm filters and injected onto a 5 μm C18 column (250×4.6 mm)on a reverse phase HPLC system. The mobile phase consisted of 69.4% 5 mMsodium phosphate buffer (pH=7.0), 30% acetonitrile, and 0.6% THF(tetrahydrofuran). The excitation wavelength was 515 nm; the emissionwavelength was 550 nm (Draper H. H. et al., Free Radic Biol Med.,15(4):353-63, 1993).

Protein determination and statistical analysis: Protein levels weredetermined by the Bradford method with Coomassie Blue (Bio-Rad)(Bradford M. M., Anal Biochem., 72:248-54, 1976). The data were given asthe mean±SD. The one-way analysis of variance test was used to analyzethe significance of the differences between the control and experimentalgroups.

This Example shows that NAC amide protects cells against glutamatetoxicity. Glutamate toxicity was evaluated by 1) morphologicalassessment of PC12 cells in the presence of glutamate; 2) measuring theamount of LDH released in the media 24 h after glutamate exposure; and3) measuring cell viability using the MTS assay. As shown in FIGS. 2A-D,cells completely lost the normal morphology of their neurites in thepresence of 10 mM glutamate, as compared to the control cells. Todetermine whether NAC amide could protect the cells from glutamatetoxicity, PC12 cells were exposed to 10 mM glutamate for 24 hours in thepresence of 750 μM NAC amide, and cell viability was examined by lightmicroscopy. The addition of NAC amide protected the PC12 cells fromglutamate toxicity by slightly decreasing the bleb formation onneurites.

To quantify the protection provided by NAC amide, PC12 cells wereexposed to 10 mM glutamate in the presence of NAC amide for 24 hours,and then the amount of LDH released was measured using the LDH assay. Asshown in FIG. 3, inclusion of 750 μM NAC amide in the assay completelyprotected the cells from cell damage, even in the presence of 10 mMglutamate (the % LDH released was 28.9±3.7%). Similar results wereobtained when cells were exposed to 10 mM glutamate in the presence ofNAC amide for 24 hours, and the cell viability was assessed by the MTSassay.

The results of Example 1 demonstrate that NAC amide treatmentsignificantly increased PC12 cell GSH levels. When cells were exposed to10 mM glutamate, a significant reduction in GSH levels was observed(Table 1).

TABLE 1 Effect of NAC amide on intracellular GSH levels in the presenceof BSO and Glutamate Group GSH Levels (nM/mg protein) Control  54 ± 13.4GLU (10 mM) * 23 ± 4.2 BSO (0.2 mM) ND NAC amide (750 μM) * 112 ± 17.8GLU + NAC amide **  88 ± 11.0 GLU + BSO + NAC amide *** 30 ± 4.3

PC12 cells were seeded and grown for 24 hours, then they were treatedwith either GLU (10 mM); NAC amide (750 μM); GLU (10 mM)+NAC amide (750μM); GLU (10 mM)+BSO (0.2 mM)+NAC amide (750 μM); or BSO (0.2 mM).Twenty hours later, cells were removed and analyzed for GSH levels, asdescribed in the text. Values represent means±SD. Statisticallydifferent values of * P<0.05 were determined, compared to control. **P<0.001 compared to glutamate-treated group. *** P<0.05 compared toglutamate-treated group. At a 750 μM concentration and 24 hour treatmenttime, NAC amide increased the PC12 cell GSH level two fold, compared tothe control group. Interestingly, similar results were obtained whenChinese hamster ovary (CHO) cells were incubated with NAC amide (datanot shown).

The intracellular levels of GSH were determined in PC12 cells incubatedwith 10 mM glutamate for 24 hours, and the effects of NAC amide wereanalyzed. Treatment of cells with NAC amide prevented the marked declineof cellular GSH levels that normally occurs after glutamate treatment(Table 1). Glutamate inhibits cystine uptake, resulting in the loss ofcellular GSH, while buthionine-sulfoximine (BSO) inhibits γ-GCS activityand thereby causes the depletion of intracellular GSH. To determinewhether the increase in intracellular GSH by NAC amide wasγ-GCS-dependent, cells were treated with 0.2 mM BSO. The simultaneoustreatment of glutamate and BSO, depleted the cell GSH to almostundetectable levels (Table 1). Interestingly, in GSH synthesis-arrestedcells, NAC amide treatment was effective and maintained 56% of thecells' GSH levels. NAC amide further protected cells againstintracellular peroxide accumulation. Malondialdehyde (MDA) is aby-product of a free radical attack on lipids. Marked increase in MDAlevels was observed in glutamate-exposed cells, as compared with thecorresponding control cells (Table 2). Treatment with NAC amidecompletely protected cells against glutamate toxicity by lowering MDAlevels.

TABLE 2 Effects of NAC amide on MDA levels in Glutamate-exposed PC12cells Group MDA Levels (nM/100 mg protein) Control 54 ± 14 GLU (10 mM)247 ± 26  NAC amide (750 μM) 81 ± 22 GLU + NAC amide 88 ± 11

Cells were plated and grown for 24 hours, and then they were exposed toglutamate (10 mM) in the presence or absence of NAC amide (750 μM).Twenty-four hours later, the cells were harvested and malondialdehydelevels were measured. Values represent means±SD. Statistically differentvalues of * P<0.002 and ** P<0.05 were determined, compared to control.*** P<0.05 compared to glutamate-treated group.

In this Example, it was determined that a high concentration ofglutamate-induced oxidative toxicity was characterized by variouspotentially detrimental changes in intracellular GSH levels, MDA levels,and LDH activity, resulting in a reduction of PC12 cell viability.Treatment with NAC amide increased intracellular GSH, and reduced MDAlevels, thereby attenuating glutamate-induced cytotoxicity. Evaluationwas done by LDH and MTS assay. Glutamate cytotoxicity has beenattributed to either excitatory action through the activation ofglutamate receptors or inhibition of cystine uptake that leads to thedecreased GSH levels. Although PC12 cells express NMDA receptors,toxicity exhibited by glutamate does not solely relate to the presenceof these receptors, as NMDA has no effect on PC12 cell death. Thedisruption of intracellular redox homeostasis by high concentrations ofglutamate is thought to be a major contributing mechanism of cellulardamage in vivo. Under conditions such as cerebral ischemia,extracellular glutamate levels increase 800%, as compared to control,which would decrease brain GSH levels by blocking cystine uptake. GSHplays an important role in antioxidant defense, and redox regulation.GSH deficiency has been associated with various neurodegenerativediseases. Intracellular GSH levels were determined by the X c- and ASCsystems. The X c-system transports cystine intracellularly in exchangefor glutamate, whereas the ASC system is a Na⁺-dependent neutral aminoacid transporter that mediates the cellular transport of cysteine.Following uptake, cystine is reduced to cysteine for intracellularglutathione synthesis. However, elevated levels of glutamate inhibitcystine uptake, and subsequent restriction of cysteine availability forthe cell, leading to GSH depletion.

In this Example, incubation of PC12 cells with glutamate resulted inreduction of GSH (Table 1) and cysteine levels (FIG. 4), when comparedto the control group. Reduced levels of cysteine indicate that thepresence of excess glutamate inhibited cystine uptake, which led todecreased GSH levels. NAC amide treatment was able to increase GSH(Table 1) and cysteine levels (FIG. 5), compared to the control group,and effectively reversed the inhibitory action of glutamate. Increasesin GSH and cysteine levels were also observed 30 minutes after NAC amidewas administered to mice. The possible mechanism for NAC amide tofacilitate the supply of cysteine may be by readily reaching the cell'sinterior, and becoming deacetylated to form cysteine. To understandwhether NAC amide could restore the GSH levels in GSH synthesis-arrestedcells, PC12 cells were incubated with glutamate (10 mM) plus BSO (0.2mM) in the presence of NAC amide (750 μM). Results showed that NAC amideelevated intracellular GSH levels in the presence of BSO, suggestingthat the effect is -GCS-independent. Therefore, NAC amide itself may actas a sulfhydryl group donor for GSH synthesis.

In summary, Example 1 shows that NAC amide protects PC12 cells againstglutamate-induced cytotoxicity by preventing glutamate-induced loss ofcellular GSH and inhibiting lipid peroxides. These studies also showthat the restoration of GSH synthesis by NAC amide in GSHsynthesis-arrested cells is -GCS-independent. Without wishing to bebound by theory, the possible mechanisms by which NAC amide can enhanceGSH are 1) supplying the rate-limiting substrate cysteine to the cellsand 2) reducing GSSG to GSH by a nonenzymatic thiol-disulfide exchange.Considering the protective effects of NAC amide againstglutamate-induced cytotoxicity, in which oxidative stress seems to beinvolved, NAC amide can play a role in the treatment ofneurodegenerative disorders such as cerebral ischemia and Parkinson'sdisease in which GSH levels are depleted in certain regions of thebrain.

Example 2

This Example examines the radioprotective effects of NAC amide. Toevaluate the protective effects of NAC amide against radiation exposure,the radioprotective role of NAC amide was compared with that of NAC withrespect to increasing the levels of GSH and returning oxidative stressparameters to their control values.

Animal studies: The irradiation of rats was performed at the RadiationOncology Department of the Phelps County Regional Medical Center inRolla, Mo., using a 16 MeV beam generated by a Varian linearaccelerator, model Clinac 1800, and in accordance with the standards ofhumane laboratory animal protocols. A 20×20 or 25×25 cm field was usedand output factors were checked once a week. Twelve animals were dividedinto 4 groups each containing 3 animals (Control, XRT, NAC amide+XRT andNAC+XRT). The radiation (XRT) control received whole body irradiation by6 Gy of 16 MeV electrons. The NAC amide+XRT group received 500 mg/kg/dayNAC amide immediately before irradiation and for three days after untilsacrifice. The rats were anesthetized and heparinized blood wascollected via cardiopuncture. Following sacrifice, liver, lung, brainand spleen were removed and stored at −70° C. until homogenization.

All experiments were performed using adult Albino SASCO Sprague Dawleyfemale rats weighing about 250 g, which were purchased from CharlesRiver Laboratories Inc. (Portage, Mich.). Twelve rats were shipped inpaper crates (4 in each crate). Rats were delivered with a certificateincluding serological, bacteriological, pathological parasitologicalinformation. They were divided into 4 cages (3 rats in each cage) andkept in a temperature controlled (20° C.) room equipped to maintain a 12h light-dark cycle. Standard rat chow (Purina rat chow) and tap waterwere supplied in individual glass bottle and given ad libitum. Water waschanged daily. Weights of the animal were taken before giving the NACamide treatment solution and amount of food eaten and water consumed wasnot measured because NAC amide was given orally but not in the drinkingwater or food.

NAC amide was provided by Novetide Ltd (Haifa Bay, Israel) includingcertificate of analysis and MSDS (lot# 40233-64). NAC amide feedingsolution was prepared freshly each day right before the administrationby weighing 1.25 g NAC amide solid sample (Type HR-120 electronicbalance, A&D Company limited, Japan. S/N: 12202464) and adding into 10ml PBS solution and put on ice. One ml of this solution wasadministrated (gavaged) per rat orally by using animal feedingbiomedical needles and 3 ml BD Luer-Lok Tip syringes. Rats receivedone-dose total-body 6Gy/16 MeV x-ray radiation and 3 rats in each groupwere held in a covered bucket and received radiation at the same time.Each day at the same time, 500 mg/Kg body weight of NAC amide wasadministrated to the animals.

All the results are normalized into values per unit (mg) of proteincontent for all the tissue samples.

Typical standard curves:

GSH:y=8.57544x−425.092,R2=0.9997

CYS:y=7.53294x+184.35,R2=0.9995

For GSH and CYS levels, 250 μL tissue homogenate was used to react with750 μL NPM solution, therefore, the total volume was 1000 μL.

As an example:

The peak area for GSH in the sample is 90860.25. The GSH concentration(nM) is calculated from the standard curve. After determining theprotein content (mg/ml) of the sample, for example: 16.5 mg/ml, thecalculation is as follows:

[(90860.25+425.092)/8.57544 nmol/L]*[1 L/1000 mL]*[1000 μL/250 μL]/(16.5mg/ml)=2.58 mmol GSH/mg protein

MDA:y=26.6869x+370.488,R2=0.9990

For MDA levels, 350 μL tissue homogenate was used to react with 100 μLof 500 ppm BHT solution and 550 μL solution of 10% TCA solution,therefore, the total volume here was 1000 μL. After boiling the wholesolution, 500 μL was taken out and react with 500 μL TBA and the totalvolume here was 1000 μL also.

As an example:

The peak area for MDA in the sample as 65289.23, The MDA concentration(nM) is calculated from the standard curve. After determining theprotein content (mg/ml) of the sample, for example: 16.5 mg/ml, theresulting calculation is as follows:

[(65289.23−370.488)/26.6869 nmol/L]*[1 L/1000 mL]*[1000 μL/350 μL]*[1000μL/500 μL]/(16.5 mg/ml)*100=84.3 nmol MDA/100 mg protein

Catalase:

Calculation for Specific Activity:

In assay solution,

k(enzyme activity)= 1/60*ln(A0/A60)*(Total Volume of reaction/volume ofsample)

A0—Absorbance at 0 secondA60—Absorbance at 60 secondIn sample, K(specific activity)=k/protein concentration.

Oxidative Stress Parameters in Animals: After the blood samples weredrawn, the animals were perfused by a cold antioxidant buffer first andthen liver, brain and kidney samples were collected aseptically, rinsedin ice-cold saline and placed in petri dishes maintained on ice. Thetissue samples kept at −70° C. for the GSH, GSSG, and MDA determinationswere made.

Glutathione (GSH) and Glutathione Disulfide (GSSG) Determination: Cellsor tissue samples were homogenized on ice and derivatized withN-(1-pyrenyl)-maleimide (NPM). The derivatized samples were injectedonto a 3 μm C18 column (Column Engineering) in a reverse phase HPLCsystem with a mobile phase of 35% water, 65% acetonitrile containing 1mL/L of acetic acid and o-phosphoric acid (R. Winters, et al., Anal.Biochem., 227:14-21 (1995) and H. H. Draper et al., Free Rad. Biol.Med., 15:353-363 (1993)). Malondialdehyde (MDA) determinations were madeas described in J. Gutteridge, Anal. Biochem., 69: 518-526 (1975).

Enzyme Activity Assays: Catalase (CAT) activity was determinedspectrophotometrically and was expressed in kunits/mg protein andkunits/10⁶ cells as described by M. Bradford, Anal. Biochem., 72:248-256(1976).

Statistical Analysis: Tabulated values represent means±standarddeviations. InStat® by GraphPad Software, San Diego, Calif. will useOne-way Analysis of Variance (ANOVA) and the Student-Newman-KeulsMultiple Comparisons Test to analyze data from experimental and controlgroups. The p values <0.05 is considered significant.

The results of the studies described in this Example are provided in thetables below. In these tables, AD4 is synonymous with NAC amide.

TABLE 3 GSH and CYS levels in BRAIN after 6Gy total-body x-ray radiationwith AD4 or NAC administration (500 mg/kg orally) GSH (nmol/mg) CYS(nmol/mg) (n = 3) level Mean SD level Mean SD CTR-1 8.19 7.5 0.7 3.614.1 0.5 CTR-2 6.75 3.88 CTR-3 7.59 4.79 XRT-1 6.42 6.6 0.3 3.48 3.8 0.5XRT-2 6.35 3.76 XRT-3 6.89 4.36 XRT + AD4-1 7.93 7.6** 0.5 4.47 4.4 0.1XRT + AD4-2 7.84 4.32 XRT + AD4-3 6.98 4.26 XRT + NAC-1 7.32 7.0 0.34.16 4.1 0.4 XRT + NAC-2 6.74 3.76 XRT + NAC-3 7.15 4.47

TABLE 4 GSH and CYS levels in LIVER after 6Gy total-body x-ray radiationwith AD4 or NAC administration (500 mg/kg orally) GSH (nmol/mg) CYS(nmol/mg) (n = 3) level Mean SD level Mean SD CTR-1 15.70 16.9 1.1 1.641.5 0.3 CTR-2 17.99 1.78 CTR-3 16.97 1.17 XRT-1 14.54 14.4* 0.2 1.34 1.40.1 XRT-2 14.26 1.39 XRT-3 14.30 1.55 XRT + AD4-1 17.45 17.2** 0.4 1.511.5 0.01 XRT + AD4-2 16.73 1.53 XRT + AD4-3 17.50 1.53 XRT + NAC-1 15.2316.3 1.0 1.25 1.5 0.2 XRT + NAC-2 16.80 1.61 XRT + NAC-3 16.93 1.51

TABLE 5 GSH and CYS levels in KIDNEY after 6Gy total-body x-rayradiation with AD4 or NAC administration (500 mg/kg orally) GSH(nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD level Mean SD CTR-1 4.625.5 0.8 10.29 11.1 0.7 CTR-2 6.25 11.56 CTR-3 5.63 11.37 XRT-1 4.91 4.80.3 8.13 8.7* 0.5 XRT-2 4.98 9.07 XRT-3 4.38 8.94 XRT + AD4-1 4.39 5.20.9 16.91 12.9** 3.4 XRT + AD4-2 6.22 11.09 XRT + AD4-3 5.02 10.81 XRT +NAC-1 5.95 6.2** 0.3 12.23 11.8** 0.7 XRT + NAC-2 6.44 12.16 XRT + NAC-36.33 11.03

TABLE 6 GSH and CYS levels in LUNG after 6Gy total-body x-ray radiationwith AD4 or NAC administration (500 mg/kg orally) GSH (nmol/mg) CYS(nmol/mg) (n = 3) level Mean SD level Mean SD CTR-1 7.04 6.2 0.7 1.911.7 0.3 CTR-2 5.87 1.85 CTR-3 5.78 1.44 XRT-1 5.24 5.1 0.8 1.26 1.6 0.3XRT-2 4.25 1.66 XRT-3 5.93 1.83 XRT + AD4-1 5.12 5.6 0.6 1.43 1.3 0.4XRT + AD4-2 5.27 1.61 XRT + AD4-3 6.28 0.91 XRT + NAC-1 5.19 5.8 1.31.16 1.9 0.7 XRT + NAC-2 7.24 2.04 XRT + NAC-3 4.95 2.43

TABLE 7 GSH and CYS levels in PLASMA after 6Gy total-body x-rayradiation with AD4 or NAC administration (500 mg/kg orally) GSH(nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD level Mean SD CTR-1 7.657.4 0.4 16.03 15.5 0.4 CTR-2 7.49 15.20 CTR-3 6.92 15.39 XRT-1 5.27 5.3*0.1 12.68 13.6* 0.9 XRT-2 5.39 13.63 XRT-3 5.31 14.45 XRT + AD4-1 7.107.6** 0.4 16.00 15.6** 0.3 XRT + AD4-2 7.44 15.45 XRT + AD4-3 7.94 15.40XRT + NAC-1 7.08 6.5**/*** 0.5 14.64 14.2*** 0.5 XRT + NAC-2 6.18 13.75XRT + NAC-3 6.27 14.36 *P < 0.05 compared to the CTR group; **P < 0.005compared to the XRT only group ***P < 0.05 compared to the XRT +AD4-treated group

TABLE 8 MDA levels in BRAIN after 6Gy total-body x-ray radiation withAD4 or NAC administration (500 mg/kg orally) MDA (nmol/100 mg) (n = 3)level Mean SD CTR-1 4.93 4.09 0.80 CTR-2 3.33 CTR-3 4.02 XRT-1 5.645.99* 0.68 XRT-2 6.76 XRT-3 5.55 XRT + AD4-1 5.79 5.48 0.33 XRT + AD4-25.53 XRT + AD4-3 5.13 XRT + NAC-1 6.42 6.15 0.72 XRT + NAC-2 6.69 XRT +NAC-3 5.33

TABLE 9 MDA levels in LIVER after 6Gy total-body x-ray radiation withAD4 or NAC administration (500 mg/kg orally) MDA (nmol/100 mg) (n = 3)level Mean SD CTR-1 4.36 4.62 0.39 CTR-2 4.44 CTR-3 5.07 XRT-1 8.9 8.36*0.53 XRT-2 8.35 XRT-3 7.83 XRT + AD4-1 4.14 4.38** 0.26 XRT + AD4-2 4.65XRT + AD4-3 4.36 XRT + NAC-1 5.1 5.07**/*** 0.04 XRT + NAC-2 5.1 XRT +NAC-3 5.02

TABLE 10 MDA levels in KIDNEY after 6Gy total-body x-ray radiation withAD4 or NAC administration (500 mg/kg orally) MDA (nmol/100 mg) (n = 3)level Mean SD CTR-1 1.61 1.69 0.09 CTR-2 1.8 CTR-3 1.68 XRT-1 2.48 2.28*0.17 XRT-2 2.17 XRT-3 2.18 XRT + AD4-1 1.5 1.64** 0.28 XRT + AD4-2 1.96XRT + AD4-3 1.45 XRT + NAC-1 1.76 1.65** 0.21 XRT + NAC-2 1.78 XRT +NAC-3 1.41

TABLE 11 MDA levels in LUNG after 6Gy total-body x-ray radiation withAD4 or NAC Administration (500 mg/kg orally) MDA (nmol/100 mg) (n = 3)level Mean SD CTR-1 1.47 1.54 0.07 CTR-2 1.53 CTR-3 1.61 XRT-1 2.3 2.80*0.45 XRT-2 2.94 XRT-3 3.17 XRT + AD4-1 1.72 1.53** 0.22 XRT + AD4-2 1.58XRT + AD4-3 1.28 XRT + NAC-1 2.58 2.52** 0.15 XRT + NAC-2 2.34 XRT +NAC-3 2.63 *P < 0.05 compared to the CTR group **P < 0.005 compared tothe XRT only group ***P < 0.05 compared to the XRT + AD4-treated group

TABLE 12 Catalase activities in KIDNEY after 6Gy total-body x-rayradiation with AD4 or NAC administration (500 mg/kg orally): Catalase(mU/mg) (n = 3) level Mean SD CTR-1 2.75 2.34 0.78 CTR-2 2.84 CTR-3 1.44XRT-1 8.73 8.69* 1.05 XRT-2 7.59 XRT-3 9.66 XRT + AD4-1 3.89 3.97** 0.56XRT + AD4-2 3.46 XRT + AD4-3 4.56 XRT + NAC-1 5.85 4.41 1.48 XRT + NAC-23.02 XRT + NAC-3 4.36

TABLE 13 Catalase activities in LUNG after 6Gy total-body x-rayradiation with AD4 or NAC administration (500 mg/kg orally): Catalase(mU/mg) (n = 3) level Mean SD CTR-1 1.50 1.24 0.33 CTR-2 0.87 CTR-3 1.37XRT-1 3.53 2.03 1.43 XRT-2 0.72 XRT-3 1.83 XRT + AD4-1 1.02 0.68** 0.29XRT + ND4-2 0.50 XRT + AD4-3 0.53 XRT + NAC-1 2.12 1.13 0.89 XRT + NAC-20.79 XRT + NAC-3 0.48

TABLE 14 Catalase activities in LIVER after 6Gy total-body x-rayradiation with AD4 or NAC administration (500 mg/kg orally). Catalase(mU/mg) (n = 3) level Mean SD CTR-1 49.48 43.03 6.13 CTR-2 42.39 CTR-337.23 XRT-1 89.10 77.44* 10.46 XRT-2 69.23 XRT-3 74.00 XRT + AD4-1 69.6359.28** 9.80 XRT + AD4-2 57.88 XRT + AD4-3 50.33 XRT + NAC-1 75.2271.11*** 3.56 XRT + NAC-2 69.09 XRT + NAC-3 69.00 *P < 0.05 compared tothe CTR group; **P < 0.05 compared to the XRT only group ***P < 0.05compared to the XRT + AD4-treated group

The data presented support the finding that NAC amide functions as astrong thiol antioxidant in radiation-induced oxidative stress. NAC doesnot increase GSH levels in tissues, presumably because it does not crossthe cell membranes. Although plasma Cys level increased significantly,this was not reflected in the liver. NAC generally provides GSH onlyduring increased demand on the GSH pool.

Upon irradiation, reactive oxygen species are formed through oxygen'sacceptance of electrons, which are involved in free radical chainreactions and are highly damaging to the cell through disruption of thecellular pro-oxidant/antioxidant balance. Normal tissue damage limitsthe radiation dose and treatment volume in radiotherapy. Radioprotectionof normal tissue by thiols offers one way in which radiation dosage canbe increased. The focus in this Example was to examine theradioprotective effects of NAC amide using a whole body radiation doseof 6 Gy, sufficient to insure that all animals should progress withlethal gastrointestinal and hematopoietic syndromes. The time pointchosen for analyses, 4 days, approximates the time that the animalswould begin to succumb to the gastrointestinal syndrome, but would beexpected to show only early changes in the hematopoietic syndrome.

GSH, a tripeptide consisting of y-glutamyl-cysteinyl-glycine, is theprinciple water-soluble intracellular free thiol and acts as aradioprotector. Several distinct mechanisms of radioprotection by GSHcan be identified and include radical scavenging, hydrogen donation todamaged molecules, reduction of peroxides, and protection of proteinthiol oxidative status. GSH has been shown to decrease in tissuesfollowing irradiation. Since GSH is an endogenous radioprotector,modification of GSH concentration may be useful as radiation protection.Cysteine provides the rate-limiting step in GSH synthesis since itsapparent Km value for γ-glutamyl-cysteine synthetase is close to theintracellular concentration of the amino acid. However, administrationof cysteine is not the ideal way to increase intracellular GSH, since itauto-oxidizes rapidly and can lead to the production of hydroxyl andthiyl radicals.

NAC, a cysteine analogue that is a mucolytic agent and a treatment forparacetamol intoxication, promotes hepatic GSH synthesis. It penetratesthe cell membrane and is rapidly deacetylated to L-cysteine, while alsostimulating GSSG reductase. NAC can rapidly increase the hepatic GSHlevels and maintain these levels for at least 6 hours (B. Wong et al.,J. Pharm. Sci., 75:878-880 (1986)). NAC has also been shown to protectChinese hamster ovary cells from lead and δ-aminolevulinic acid-inducedtoxicity through restoration of the oxidative status of the cells by GSHreplenishment. It has been demonstrated that NAC protects liver andbrain of C57BL/6 mice from GSH depletion as a result of lead poisoning.Radioprotective effects of select thiols such as indomethacin, WR-2721,cysteamine, and diethyldithiocarbamate have been reported, though athigher concentrations these induce cellular toxicity. Theradioprotective effect of NAC has been demonstrated in humangranulocyte/macrophage-colony forming cells. However, it has also beenshown that the more radioresistant SW-1573 human squamous lung carcinomacell line was not protected from X-ray induced cell death by NAC. NACamide is more lipophilic and able to more easily cross cell membranesthan NAC. In this Example, the radioprotective function of NAC amide wascompared with that of NAC in terms of increasing GSH levels andreturning oxidative stress parameters to their control values.

The exposure of membrane lipids to reactive oxygen species such as thehydroxyl radical can initiate a chain reaction in polyunsaturated fattyacid moieties, which results in peroxidation and causes degradation ofmembrane function. MDA is a degradation product of the highly unstablelipid peroxides. As observed in this Example, irradiation of SpragueDawley rats resulted in increased MDA levels in liver and lung. Upontreatment with NAC amide concurrent with irradiation, lung MDA levelswere significantly lowered, while treatment with NAC did not change theMDA levels significantly.

It is generally accepted in the field of radiobiology that the mechanismof individual cell killing by radiation exposure is due to direct andindirect ionizing effects specifically upon DNA in the cell nucleus,although it becomes apparent that in a complex organism there are ROSeffects of some potential importance on membrane lipids and proteins aswell as on nucleic acids. Furthermore, acute whole body irradiation ofthe intact animal under conditions modeling the so called“gastrointestinal syndrome” causes changes in several tissues apart fromgastrointestinal tract, and some of these effects can be ameliorated bythe use of NAC amide. A given syndrome such as the “gastrointestinalsyndrome” can actually involve a complex of changes in multiple tissuesand organs. Radiation pneumonitis can be a serious hazard in thetherapeutic irradiation of patients with lung cancer. NAC amide may beconsidered for use as a thiol radioprotectant to protect against such acomplication. Thus, in accordance with the invention, NAC amidesignificantly increases thiol levels in plasma and liver and performsbetter than NAC as a radioprotecting agent.

Example 3

This Example describes a treatment regimen suitable for humans. NACamide is administered between 1 and three grams per day, in two divideddoses, between meals (on an empty stomach). Encapsulated NAC amide (aformulation of NAC amide comprising 500 mg NAC amide and optionally, 250mg USP grade crystalline ascorbic acid, and not more than 0.9 mgmagnesium stearate, NF grade in an OO-type gelatin capsule) is suitablefor administration. The administration of exogenous NAC amide isexpected to provide a dose response effect in patients, despite theproduction of large quantities of glutathione in the human body.

Example 4

This Example describes a combination pharmaceutical composition toameliorate the detrimental effects of acetaminophen, a drug thatconsumes glutathione in the liver during metabolism and, in excessdoses, causes liver damage due to oxidative damage. The compositionincludes 500 mg NAC amide, 250 mg crystalline ascorbic acid and 350 mgacetaminophen.

Example 5

This Example describes a combination pharmaceutical composition toameliorate the detrimental effects of chlorpromazine, a phenothiazinedrug that causes side effects, including tardive dyskinesia, which maybe associated with excess free radical reactions. The compositionincludes 500 mg NAC amide, 250 mg crystalline ascorbic acid and 200 mgchlorpromazine.

Example 6

This Example describes a combination pharmaceutical composition toameliorate the detrimental effects of aminoglycoside drugs(antibiotics), nonlimiting examples of which include neomycin,kanamycin, amikacin, streptomycin, gentamycin, sisomicin, netilmicin andtobramycin, a drug class which may be associated with varioustoxicities. This damage may be related to oxidative damage orconsumption of glutathione during metabolism. The composition accordingto the present invention is an intravenous formulation, including theaminoglycoside in an effective amount, and NAC amide in an amount ofabout 10-20 mg/kg. Ascorbic acid in an amount of 5-10 mg/kg may be addedas a stabilizer.

Example 7

This Example describes a urethral insert comprising NAC amide. Acomposition containing 200 mg NAC amide, 50 mg ascorbic acid per unitdosage is mixed with carageenan and/or agarose and water in aquick-gelling composition, and permitted to gel in a cylindrical formhaving a diameter of about 3 mm and a length of about 30 mm. Thecomposition is subjected to nitric oxide to cause between 0.1-10% of theNAC amide to be converted to nitroso-NAC amide. The gelled agarose isthen freeze dried under conditions that allow shrinkage. Thefreeze-dried gel is than packaged in a gas barrier package, such as afoil pouch or foil “bubble-pack”. The freeze-dried gel may then be usedas a source of nitroso-NAC amide for administration transmucosally. Thecylindrical freeze-dried gel may be inserted into the male urethra fortreatment of impotence, or administered sublingually for systemicvasodilation.

Example 8

This Example describes an oral formulation for prophylaxis of vasculardisease, e.g., in men over 40. The composition includes 500 mg NACamide, 250 mg USP grade crystalline ascorbic acid and 50 mg USP acetylsalicylic acid (aspirin) in an OO-type gelatin capsule. Typicaladministration is twice per day. The acetyl salicylic acid may beprovided in enteric release pellets within the capsule to retardrelease.

Example 9

This Example describes an oral formulation for prophylaxis of vasculardisease. The composition contains 500 mg NAC amide, 200 mg USP gradecrystalline ascorbic acid, and 200 mg arginine in an OO-type gelatincapsule. Arginine is the normal starting substrate for the production ofnitric oxide. Because arginine is normally in limited supply, a relativedeficiency of arginine may result in impaired vascular endothelialfunction.

Example 10

This Example describes an oral formulation for prophylaxis of vasculardisease. The composition includes 500 mg NAC amide, 200 mg USP gradecrystalline ascorbic acid, and 200 mg vitamin E succinate in an OO-typegelatin capsule. Vitamin E consumption reduces the risk of heart attackand other vascular disease. Vitamin E succinate (alpha-tocopherolsuccinate) is a dry powder.

Example 11

This Example describes an oral formulation for prophylaxis of vasculardisease. Nonspecific esterases having broad substrate specificity arepresent in the plasma. According to the present invention, esters areformed between agents that are useful combination therapies in order toprovide for efficient administration, high bioavailability, andpharmaceutical stability. Preferred esters include alphatocopherol-ascorbate, alpha tocopherol-salicylate, andascorbyl-salicylate. The tocopherol ester maintains the molecule in areduced state, allowing full antioxidant potential after ester cleavage.These esters may be administered alone or in combination with otheragents, for example NAC amide. Typically, the esters are administered todeliver an effective dose of salicylate equivalent of 100 mg per day forprophylaxis, or 750-1000 mg per dose for treatment of inflammatorydiseases. Tocopherol is administered in an amount of 100-500 IUequivalent. Ascorbate is administered in an amount of up to 1000 mgequivalent. In order to enhance availability, a non-specific esterasemay be provided in the formulation to cleave the ester after dissolutionof the capsule. Therefore, a non-specific esterase, such as a bacterialor saccharomyces (yeast) enzyme, or an enriched enzyme preparation, maybe included in the formulation as a powder or as pellets in the capsule.

Example 12

This Example describes an oral formulation for prophylaxis of vasculardisease. The composition includes 500 mg reduced NAC amide, 200 mg USPgrade crystalline ascorbic acid, and 100 mg nordihydroguaretic acid, inan OO-type gelatin capsule. Typical administration is twice per day.Nordihydroguaretic acid is a known lipoxygenase inhibitor. Thus, thiscomposition may be used to treat inflammatory processes or asprophylaxis against vascular disease.

Example 13

This Example describes a study observing the survival of rats receivingwhole body, single-dose irradiation by X-rays (XRT) in the presence orabsence of NAC or NAC amide (TOVA). In this experiment, thirty-ninefemale Sprague-Dawley rats ranging from about 150-200 g were subjectedto total body, single-dose X-ray irradiation (9 Gy, 16 Mev). The samegroups were designated to receive either NAC or TOVA. For thepre-treatment groups (n=6 in each group), the first treatment of NAC orTOVA was administered 30 minutes to 1 hour before irradiation. For thepost-pretreatment groups (n=6 in each group), the first treatment of NACor TOVA was administered 30 minutes to 1 hour after the irradiation. Forgroups receiving NAC or TOVA, the same amount (500 mg/kg NAC or TOVAdaily) was administered for 4 or 5 consecutive days.

Group 1 was a control group (n=3), where rats received the same amountof saline solution daily for 5 consecutive days without XRT. Group 2rats received NAC only (n=3) at an amount of 500 mg/kg body weight NACdaily for 5 consecutive days without XRT. Group 3 rats received TOVAonly (n=3) at an amount of 500 mg/kg body weight TOVA daily for 5consecutive days without XRT. Group 4 rats received radiation (XRT) only(n=6) and received the same amount of saline solution daily for 5consecutive days after single dose total-body XRT irradiation.

Group 5 rats received one treatment of NAC at 500 mg/kg body weightbefore XRT (XRT+NAC pre-treated), which was then followed by 500 mg/kgbody weight NAC daily for 4 consecutive days after XRT. Group 6 ratsreceived XRT, followed by daily doses of NAC at 500 mg/kg body weightfor 5 consecutive days after XRT (XRT+NAC post-treated). Group 7 ratsreceived one treatment of NAC at 500 mg/kg body weight before XRT(XRT+TOVA pre-treated), which was then followed by 500 mg/kg body weightTOVA daily for 4 consecutive days after XRT. Group 8 rats received XRT,followed by daily doses of TOVA at 500 mg/kg body weight for 5consecutive days after XRT (XRT+TOVA post-treated). All rats were thengiven a normal diet post-treatment.

The rats were observed twice a day, and the survival status of rats ineach group will be recorded. The mean survival days were calculated foreach group and compared to the survival differences of the three groupsof rats at the end of the experiment. The radioprotective effects of NACand TOVA treatment on the survival of those irradiated rats were thenevaluated, as shown in the following tables.

Table 15 shows the number of animals that survived under conditionswhere NAC or TOVA was administered pre- or post-XRT treatment.

# of # of percentage animals animals survival survival Groups # ofanimals dead survived rate rate XRT only (n = 6)-1st time 2 4 (4 +2)/(6 + 6)  50% (n = 6)-2nd time 4 2 XRT + NAC (pre-treated) (n = 6)-1sttime 1 5 (5 + 5)/(6 + 6) 83.3%  (n = 6)-2nd time 1 5 XRT + TOVA(pre-treated) (n = 6)-1st time 0 6 (6 + 6)/(6 + 6) 100% (n = 6)-2nd time0 6 Control (no XRT and any (n = 3)-1st time 0 3 (3 + 3)/(3 + 3) 100%treatment) (n = 3)-2nd time 0 3 NAC only (n = 2)-2nd time 0 2 (2)/(2)100% TOVA only (n = 3)-2nd time 0 3 (3)/(3) 100% XRT + NAC(post-treated) (n = 6)-2nd time 4 2 (2)/(6) 33.3%  XRT + TOVA(post-treated) (n = 6)-2nd time 2 4 (4)/(6) 66.7% 

Table 16 shows the survival rate percentage of rats receiving NAC orTOVA pre- or post-XRT treatment.

Groups percentage survival rate XRT only  50% XRT + NAC(pre-treated)83.3%  XRT + TOVA(pre-treated) 100% Control (no XRT and any treatment)100% NAC only 100% TOVA only 100% XRT + NAC(post-treated) 33.3%  XRT +TOVA(post-treated) 66.7% 

FIG. 6 is a graphical representation comparing the percentage survivalrates as presented in Table 16. These results show that rats pre-treatedwith NAC or TOVA before XRT have a higher survival rate than thosereceiving XRT alone.

All patent applications, published applications, patents, texts, andliterature references cited in this specification are herebyincorporated herein by reference in their entirety.

As various changes can be made in the above methods and compositionswithout departing from the scope and spirit of the invention asdescribed, it is intended that all subject matter contained in the abovedescription, shown in the accompanying drawings, or defined in theappended claims be interpreted as illustrative, and not in a limitingsense.

1. A pharmaceutical composition for increasing glutathione levels toreduce overproduction of oxidants in cells and tissues, comprisingN-acetylcysteine amide (NAC amide), or a pharmaceutically acceptablesalt, ester, or derivative thereof.
 2. A method of increasingantioxidant levels in cells and tissues of an organism, comprisingadministering NAC amide to the organism in an amount effective toincrease antioxidant levels.
 3. The method according to claim 2, whereinsaid organism is a human being suffering from a condition, disease,disorder, or pathology associated with over production of oxidants. 4.The method according to claim 3, wherein said condition, disease,disorder, or pathology is selected from the group consisting of AIDS,diabetes, macular degeneration, congestive heart failure, cardiovasculardisease, coronary artery restenosis, lung disease, inflammatory disease,asthma, RNA virus infection, DNA virus infection, sepsis, sepsis,osteoporosis, bone disease, infection by microorganisms, toxin exposure,radiation exposure, burn trauma, prion disease, neurological disease,blood disease, blood cell disease, arterial disease and muscle disease.5. The method according to claim 4, wherein the condition, disease,disorder, or pathology is malaria.
 6. The method according to claim 4,wherein the condition, disease, disorder, or pathology is tuberculosis.7. The method according to claim 4, wherein the blood disease is sicklecell anemia.
 8. A method of protecting an organism fromradiation-induced oxidative stress, comprising administering aradioprotective amount of NAC amide or a derivative of NAC amide to theorganism.
 9. The method according to claim 8, wherein NAC amide orderivative of NAC amide is administered orally in an amount of 500mg/kg.
 10. A method of increasing levels of thiol antioxidants in anorganism, comprising administering NAC amide or derivative of NAC amideto the organism in an amount effective to increase thiol antioxidantlevels.
 11. The method according to claim 10, wherein the thiolantioxidant is glutathione or cysteine.
 12. The method according toclaim 10, wherein the levels of thiol antioxidant increase in liver andplasma.
 13. The method according to claim 10, wherein NAC amide orderivative of NAC amide is administered orally in an amount of 500mg/kg.
 14. A method of killing or inhibiting the growth of bacteria incells of an infected host, comprising: providing NAC amide or derivativeof NAC amide in an amount effective to induce the production of HIF-1 orHIF-1 α in white blood cells of the host, thereby enhancing the capacityof the white blood cells to kill or inhibit the growth of themicroorganisms.
 15. A method of blocking the effects of Rac1b-inducedROS production associated with metalloproteinase activity, comprising:administering or introducing NAC amide or derivative of NAC amide tocells, tissues, and/or a subject in need thereof, thereby targetingmolecules in the pathway leading to tissue damage and degradation.
 16. Amethod of blocking or inhibiting the effects of MMP-3 metalloproteinaseon Rac1b-induced ROS production, comprising: administering orintroducing NAC amide or derivative of NAC amide to cells, tissues,and/or a subject in need thereof, to block or inhibit the activity ofMMP-3 which leads to tissue damage and degradation.
 17. A method ofstimulating endogenous production of cytokines and hematopoieticfactors, comprising: administering or introducing NAC amide orderivative of NAC amide to cells, tissues, and/or a subject in needthereof for a period of time to stimulate the endogenous production toobtain a pre-determined, desired therapeutic effect.
 18. A method ofdetecting NAC-amide responsive changes in gene expression in a cell,tissue, and/or a subject, comprising: administering or introducing NACamide or derivative of NAC amide to the cell, tissue, and/or subject fora period of time to induce changes in gene expression and detecting thechanges in gene expression.
 19. The method of claim 18, wherein cell isan endothelial cell.
 20. The method of claim 18, wherein the tissue isvascular tissue.
 21. The method of claim 18, wherein the changes in geneexpression are detected by microarray analysis, RT-PCR, NorthernBlotting, immunofluorescence, immunoblotting, or enzyme-linkedimmunosorbent assay.
 22. The method according to claim 18, comprisingadministering or providing NAC amide or derivative of NAC amide coupledto nanoparticles.
 23. A method of directed delivery of NAC amide orderivative of NAC amide to host cells expressing high levels of surfacereceptor for a ligand, comprising: a) coupling NAC amide or derivativeof NAC amide to the surface receptor ligand to form a NAC amide-ligandconjugate; b) adsorbing the NAC amide-ligand conjugate ontonanoparticles; and c) introducing the nanoparticles of step (b) into thehost.
 24. A method of directed delivery of NAC amide or derivative ofNAC amide to host cells expressing high levels of surface receptor for aligand, comprising: a) conjugating acetylated dendritic nanopolymers toa ligand; b) coupling the conjugated ligand of step (a) to NAC amide orderivative of NAC amide to form NAC amide-ligand nanoparticles; and c)introducing the nanoparticles of step (b) into the host.
 25. The methodaccording to claim 23 or claim 24, wherein the ligand is folic acid orglutathione.
 26. The method according to claim 23 or claim 24, whereinthe nanoparticles are PAMAM dendritic polymers.
 27. A compound offormula I:

wherein: R₁ is OH, SH, or S—S-Z; X is C or N; Y is NH₂, OH, CH₃—C═O, orNH—CH₃; R₂ is absent, H, or ═O R₃ is absent or

wherein: R₄ is NH or O; R₅ is CF₃, NH₂, or CH₃ and wherein: Z is

with the proviso that if R₁ is S—S-Z, X and X′ are the same, Y and Y′are the same, R₂ and R₆ are the same, and R₃ and R₇ are the same. 28.The compound of claim 27, wherein R₁ is S, X is C, Y is NH₂, R₂ is ═O,R₃ is

R₄ is O, and R₅ is CH₃.
 29. The compound of claim 28, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 30. Thecompound of claim 27, wherein R₁ is S, X is C, Y is NH—CH₃, R₂ is H, R₃is

R₄ is O, and R₅ is CH₃.
 31. The compound of claim 30, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 32. Thecompound of claim 27, wherein R₁ is S, X is N, Y is CH₃—C═O, R₂ is H,and R₃ is absent.
 33. The compound of claim 27, wherein R₁ is S, X is C,Y is NH₂, R₂ is ═O, R₃ is

R₄ is O, and R₅ is CF₃.
 34. The compound of claim 33, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 35. Thecompound of claim 27, wherein R₁ is O, X is C, Y is NH₂, R₂ is ═O, R₃ is

R₄ is O, and R₅ is CH₃.
 36. The compound of claim 35, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 37. Thecompound of claim 27, wherein R₁ is S, X is C, Y is OH, R₂ is absent, R₃is

R₄ is O, and R₅ is CH₃.
 38. The compound of claim 37, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 39. Thecompound of claim 27, wherein R₁ is S, X is C, Y is NH₂, R₂ is ═O, R₃ is

R₄ is NH, and R₅ is NH₂.
 40. The compound of claim 39, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 41. Thecompound of claim 27, wherein R₁ is O, X is C, Y is OH, R₂ is absent, R₃is

R₄ is O, and R₅ is CH₃.
 42. The compound of claim 41, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 43. Thecompound of claim 27, wherein R₁ is S—S-Z, X is C, Y is NH₂, R₂ is ═O,R₃ is

R₄ is O and R₅ is CH₃.
 44. The compound of claim 43, wherein thecompound is chiral and is selected from the group consisting of aD-isomer, a L-isomer, and a racemic mixture of D- and L-isomers.
 45. Aprocess for preparing an L-isomer of the compound of claim 27,comprising: (a) adding a base to L-cystine diamide dihydrochloride toproduce a first mixture, and subsequently heating the first mixtureunder vacuum; (b) adding a methanolic solution to the heated firstmixture; (c) acidifying the mixture with alcoholic hydrogen chloride toobtain a first residue; (d) dissolving the first residue in a firstsolution comprising methanol saturated with ammonia; (e) adding a secondsolution to the dissolved first residue to produce a second mixture; (f)precipitating and washing the second mixture; (g) filtering and dryingthe second mixture to obtain a second residue; (h) mixing the secondresidue with liquid ammonia and an ethanolic solution of ammoniumchloride to produce a third mixture; and (i) filtering and drying thethird mixture, thereby preparing the L-isomer compound.
 46. The processof claim 45, wherein the base comprises liquid ammonia or methylamine.47. The process of claim 45, wherein the second solution compriseswater, an acetate salt, and an anhydride.
 48. The process of claim 47,wherein the acetate salt comprises sodium acetate or sodiumtrifluoroacetate.
 49. The process of claim 47, wherein the anhydridecomprises acetic anhydride or trifluoroacetic anhydride.
 50. The processof claim 45, wherein the second solution comprises dichloromethane,triethylamine, and 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.51. The process of claim 45, wherein step (h) further comprises mixingthe second residue in the presence of sodium metal.
 52. The process ofclaim 45, further comprising the steps of (j) dissolving the L-isomercompound in ether; (k) adding to the dissolved L-isomer compound anethereal solution of lithium aluminum hydride, ethyl acetate, and waterto produce a fourth mixture; and (l) filtering and drying the fourthmixture, thereby preparing the L-isomer compound.
 53. A process forpreparing an D-isomer of the compound of claim 27, comprising: (a)adding a base to D-cystine diamide dihydrochloride to produce a firstmixture, and subsequently heating the first mixture under vacuum; (b)adding a methanolic solution to the heated first mixture; (c) acidifyingthe mixture with alcoholic hydrogen chloride to obtain a first residue;(d) dissolving the first residue in a first solution comprising methanolsaturated with ammonia; (e) adding a second solution to the dissolvedfirst residue to produce a second mixture; (f) precipitating and washingthe second mixture; (g) filtering and drying the second mixture toobtain a second residue; (h) mixing the second residue with liquidammonia, sodium metal, and an ethanolic solution of ammonium chloride toproduce a third mixture; and (i) filtering and drying the third mixture,thereby preparing the L-isomer compound.
 54. The process of claim 53,wherein the base comprises liquid ammonia or methylamine.
 55. Theprocess of claim 53, wherein the second solution comprises water, anacetate salt, and an anhydride.
 56. The process of claim 55, wherein theacetate salt comprises sodium acetate or sodium trifluoroacetate. 57.The process of claim 55, wherein the anhydride comprises aceticanhydride or trifluoroacetic anhydride.
 58. The process of claim 53,wherein the second solution comprises dichloromethane, triethylamine,and 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
 59. Theprocess of claim 53, wherein step (h) further comprises mixing thesecond residue in the presence of sodium metal.
 60. The process of claim53, further comprising the steps of (j) dissolving the D-isomer compoundin ether; (k) adding to the dissolved D-isomer compound an etherealsolution of lithium aluminum hydride, ethyl acetate, and water toproduce a fourth mixture; and (l) filtering and drying the fourthmixture, thereby preparing the D-isomer compound.
 61. A process forpreparing an L-isomer of the compound of claim 27, comprising: (a)mixing S-benzyl-L-cysteine methyl ester hydrochloride orO-benzyl-L-serine methyl ester hydrochloride with a base to produce afirst mixture; (b) adding ether to the first mixture; (c) filtering andconcentrating the first mixture; (d) repeating steps (c) and (d), toobtain a first residue; (e) adding ethyl acetate and a first solution tothe first residue to produce a second mixture; (f) filtering and dryingthe second mixture to produce a second residue; (g) mixing the secondresidue with liquid ammonia, sodium metal, and an ethanolic solution ofammonium chloride to produce a third mixture; and (h) filtering anddrying the third mixture, thereby preparing the L-isomer compound. 62.The process of claim 61, wherein the base comprises a methanolicsolution of ammonia or methylamine.
 63. The process of claim 61, whereinthe second solution comprises water, an acetate salt, and an anhydride.64. The process of claim 63, wherein the acetate salt comprises sodiumacetate or sodium trifluoroacetate.
 65. The process of claim 63, whereinthe anhydride comprises acetic anhydride or trifluoroacetic anhydride.66. The process of claim 61, wherein the second solution comprisesdichloromethane, triethylamine, and1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
 67. The process ofclaim 61, further comprising the steps of (j) dissolving the L-isomercompound in ether; (k) adding to the dissolved L-isomer compound anethereal solution of lithium aluminum hydride, ethyl acetate, and waterto produce a fourth mixture; and (l) filtering and drying the fourthmixture, thereby preparing the L-isomer compound.
 68. A process forpreparing an D-isomer of the compound of claim 27, comprising: (a)mixing S-benzyl-D-cysteine methyl ester hydrochloride orO-benzyl-D-serine methyl ester hydrochloride with a base to produce afirst mixture; (b) adding ether to the first mixture; (c) filtering andconcentrating the first mixture; (d) repeating steps (c) and (d), toobtain a first residue; (e) adding ethyl acetate and a first solution tothe first residue to produce a second mixture; (f) filtering and dryingthe second mixture to produce a second residue; (g) mixing the secondresidue with liquid ammonia, sodium metal, and an ethanolic solution ofammonium chloride to produce a third mixture; and (h) filtering anddrying the third mixture, thereby preparing the D-isomer compound. 69.The process of claim 68, wherein the base comprises a methanolicsolution of ammonia or methylamine.
 70. The process of claim 68, whereinthe second solution comprises water, an acetate salt, and an anhydride.71. The process of claim 70, wherein the acetate salt comprises sodiumacetate or sodium trifluoroacetate.
 72. The process of claim 70, whereinthe anhydride comprises acetic anhydride or trifluoroacetic anhydride.73. The process of claim 68, wherein the second solution comprisesdichloromethane, triethylamine, and1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
 74. The process ofclaim 68, further comprising the steps of (j) dissolving the D-isomercompound in ether; (k) adding to the dissolved D-isomer compound anethereal solution of lithium aluminum hydride, ethyl acetate, and waterto produce a fourth mixture; and (l) filtering and drying the fourthmixture, thereby preparing the D-isomer compound.
 75. A process forpreparing a compound of claim 27, comprising: (a) mixing cystaminedihydrochloride with ammonia, water, sodium acetate, and aceticanhydride to produce a first mixture; (b) allowing the first mixture toprecipitate; (c) filtering and drying the first mixture to produce afirst residue; (d) mixing the second residue with liquid ammonia, sodiummetal, and an ethanolic solution of ammonium chloride to produce asecond mixture; (e) filtering and drying the second mixture, therebypreparing the compound.
 76. A NAC amide compound or derivative selectedfrom the group consisting of:


77. A food additive comprising NAC amide or a NAC amide derivative ofclaim 76.